HCV polymerase inhibitors

ABSTRACT

The invention provides compounds of the formula: 
                         
wherein B is a nucleobase selected from the groups (a) to (d):
 
                         
and the other variables are as defined in the claims,
 
which are of use in the treatment or prophylaxis of hepatitis C virus infection, and related aspects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Nonprovisional application is a Continuation application ofapplication Ser. No. 15/210,500 filed on Jul. 14, 2016, now U.S. Pat.No. 9,540,411, which is a Continuation of application Ser. No.14/634,449 filed on Feb. 27, 2015, now U.S. Pat. No. 9,481,703, which isa Continuation-In-Part application of PCT International Application No.PCT/SE2014/051005 filed on Sep. 2, 2014, which claims priority under 35U.S.C. §119 on Patent Application No. 1351026-8 filed in Sweden on Sep.4, 2013, on Patent Application No. 1351169-6 filed in Sweden on Oct. 3,2013, and on Patent Application No. 1450152-2 filed in Sweden on Feb.12, 2014, the entire contents of each of which are hereby incorporatedby reference.

TECHNICAL FIELD

The present invention relates to nucleoside derivatives which areinhibitors of the polymerase of hepatitis C virus (HCV). The inventionfurther relates to prodrugs of the nucleoside derivatives, compositionscomprising them, and methods for their use in the treatment orprophylaxis of HCV infection.

BACKGROUND OF THE INVENTION

HCV is a single stranded, positive-sense RNA virus belonging to theFlaviviridae family of viruses in the hepacivirus genus. The NS5B regionof the RNA polygene encodes an RNA dependent RNA polymerase (RdRp),which is essential to viral replication. Following the initial acuteinfection, a majority of infected individuals develop chronic hepatitisbecause HCV replicates preferentially in hepatocytes but is not directlycytopathic. In particular, the lack of a vigorous T-lymphocyte responseand the high propensity of the virus to mutate appear to promote a highrate of chronic infection. Chronic hepatitis can progress to liverfibrosis, leading to cirrhosis, end-stage liver disease and HCC(hepatocellular carcinoma), making it the leading cause of livertransplantations.

There are six major HCV genotypes and more than 50 subtypes, which aredifferently distributed geographically. HCV genotype 1 is thepredominant genotype in Europe and in the U.S. The extensive geneticheterogeneity of HCV has important diagnostic and clinical implications,perhaps explaining difficulties in vaccine development and the lack ofresponse to current therapy.

Transmission of HCV can occur through contact with contaminated blood orblood products, for example following blood transfusion or intravenousdrug use. The introduction of diagnostic tests used in blood screeninghas led to a downward trend in post-transfusion HCV incidence. However,given the slow progression to the end-stage liver disease, the existinginfections will continue to present a serious medical and economicburden for decades.

The first generation HCV therapies were based on (pegylated)interferon-alpha (IFN-α) in combination with ribavirin. This combinationtherapy yields a sustained virologic response in more than 40% ofpatients infected by genotype 1 viruses and about 80% of those infectedby genotypes 2 and 3. Beside the limited efficacy on HCV genotype 1,this combination therapy has significant side effects and is poorlytolerated in many patients. Major side effects include influenza-likesymptoms, hematologic abnormalities and neuropsychiatric symptoms. Thesecond generation of HCV treatments added the HCV protease inhibitorstelepravir or boceprevir, allowing treatment times to be shortened, butgenerating a significant number of serious side-effects. A majorimprovement in treatment was possible with the introduction of theprotease inhibitor simeprevir and the HCV polymerase inhibitorsofosbuvir. These were initially co-administered with interferon andribavirin, but more recently the co-administration of simeprevir(WO2007/014926) and sofosbuvir (WO2008/121634) has allowedinterferon-free and ribavirin-free HCV treatment with further diminishedtreatment times and dramatically decreased side effects.

An advantage of nucleoside/nucleotide HCV polymerase inhibitors such assofosbuvir, is that they tend to be active against several of the HCVgenotypes. Sofosbuvir for example has been approved by the FDA and EMAfor treatment of HCV genotypes 1 and 4. However, in the Fission phaseIII clinical trials reported in Lawitz et al, N. Eng. J. Med. 2013;368:1878-87, it was noted “Response rates in the sofosbuvir—ribaviringroup were lower among patients with genotype 3 infection than amongstthose with genotype 2 infection (56% vs. 97%)”. Hence there is a needfor more effective, convenient and better-tolerated treatments.

Experience with HIV drugs, in particular with HIV protease inhibitors,has taught that sub-optimal pharmacokinetics and complex dosing regimesquickly result in inadvertent compliance failures. This in turn meansthat the 24 hour trough concentration (minimum plasma concentration) forthe respective drugs in an HIV regime frequently falls below the IC₉₀ orED₉₀ threshold for large parts of the day. It is considered that a 24hour trough level of at least the IC₅₀, and more realistically, the IC₉₀or ED₉₀, is essential to slow down the development of drug escapemutants. Achieving the necessary pharmacokinetics and drug metabolism toallow such trough levels provides a stringent challenge to drug design.

The NS5B RdRp is absolutely essential for replication of thesingle-stranded, positive sense HCV RNA genome which makes it anattractive target for the development of antiviral compounds. There aretwo major classes of NS5B inhibitors: non-nucleoside inhibitors (NNIs)and nucleoside analogues. The NNIs bind to allosteric regions of theprotein whereas the nucleoside inhibitors are anabolized to thecorresponding nucleotide and act as alternative substrate for thepolymerase. The formed nucleotide is then incorporated in the nascentRNA polymer chain and can terminate the growth of the polymer chain. Todate, bath nucleoside and non-nucleoside inhibitors of NS5B are known.

As stated above, the inhibition mechanism of nucleoside inhibitorsinvolves phosphorylation of the nucleoside to the correspondingtriphosphate. The phosphorylation is commonly mediated by host cellkinases and is an absolute requirement for the nucleoside to be activeas an alternative substrate for the NS5B polymerase. Typically, thefirst phosphorylation step, i.e. conversion of the nucleoside to thenucleoside 5′-monophosphate is the rate limiting step. Subsequentconversion of the monophosphate to the di- and tri-phosphate usuallyproceed facile and are usually not rate limiting. A strategy forincreasing nucleoside triphosphate production is to use cell permeablenucleoside prodrugs of the monophosphate, i.e. a nucleoside carrying amasked phosphate moiety, a “prodrug moiety”, which are susceptible tointracellular enzymatic activation leading to a nucleosidemonophosphate. The thus formed monophosphate is subsequently convertedto the active triphosphate by cellular kinases.

Chemical modifications of an active compound to afford a potentialprodrug produces an entirely new molecular entity which can exhibitundesirable physical, chemical and biological properties, thus theidentification of optimal prodrugs remains an uncertain and challengingtask.

There is a need for HCV inhibitors that may overcome the disadvantagesof current HCV therapy such as side effects e.g. toxicity, limitedefficacy, lack of pan-genotypic coverage, the emerging of resistance,and compliance failures, as well as improve the sustained viralresponse.

The present invention provides new HCV inhibiting compounds which haveuseful properties regarding one or more of the following parameters:antiviral efficacy; pan-genotypic coverage; favourable profile ofresistance development; lack of toxicity and genotoxicity; favourablepharmacokinetics and pharmacodynamics; and ease of formulation andadministration. The skilled person will appreciate that an HCVinhibiting compound of the present invention need not demonstrate animprovement in every respect over all known compounds but may insteadprovide a balance of properties which in combination mean that the HCVinhibiting compound is a valuable alternative pharmaceutical agent.

Compounds of the invention may also be attractive due to the fact thatthey lack activity against other viruses, i.e. are selective, inparticular against HIV. HIV infected patients often suffer fromco-infections such as HCV. Treatment of such patients with HCV inhibitorthat also inhibits HIV may lead to the emergence of resistant HIVstrains.

DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides compounds represented byformula I:

wherein;B is a nucleobase selected from the groups (a) to (d):

wherein Y is N or —C(R¹⁹)—;

-   R¹ is H, C(═O)R³⁰, C(═O)CHR³¹NH₂, CR³²R^(32′)OC(═O)CHR³³NH₂, or R¹    is selected from the groups (i) to (vi):

-   R² is H, C(═O)R³⁰, C(═O)CHR³¹NH₂, CR³²R^(32′)OC(═O)CHR³³NH₂ or    CR³²R^(32′)OC(═O)R³⁰; or R¹ and R² together form a bivalent linker    of formula:

-   R³ is OH, C₁-C₆alkoxy, C₃-C₇cycloalkoxy, C₃-C₇cycloalkylC₁-C₃alkoxy,    benzyloxy, O—(C₁-C₆alkylene)-T-R²¹ or NHC(R¹⁵)(R^(15′))C(═O)R¹⁶;-   R⁴, R⁵, R⁷ and R⁸ are each independently H, C₁-C₆alkyl,    C₁-C₆haloalkyl, C₁-C₆hydroxyalkyl, halo, —OR¹⁸, —SR¹⁸ or —N(R¹⁸)₂;-   R⁶, R⁹, R¹⁰, R¹¹ are each independently selected from H, C₁-C₆alkyl,    C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl, C₁-C₆haloalkyl,    C₁-C₆hydroxyalkyl, halo, OR¹⁸, SR¹⁸, N(R¹⁸)₂, —NHC(O)OR¹⁸,    —NHC(O)N(R¹⁸)₂, —CN, —NO₂, —C(O)R¹⁸, —C(O)OR¹⁸, —C(O)N(R¹⁸)₂ and    —NHC(O)R¹⁸, wherein said C₂-C₆alkenyl group and said C₂-C₆alkynyl    group can be optionally substituted with halo or C₃-C₅cycloalkyl;-   R¹² is H or —(C₁-C₆alkylene)-T-R²¹, phenyl, indolyl or naphthyl    which phenyl, indolyl or naphthyl group is optionally substituted    with 1, 2 or 3 substituents each independently selected from halo,    C₁-C₆alkyl, C₂-C₆alkenyl, C₁-C₆haloalkyl, hydroxyC₁-C₆alkyl,    C₃-C₆cycloalkyl, C₁-C₆alkylcarbonyl, C₃-C₆cycloalkylcarbonyl    C₁-C₆alkoxy, C₁-C₆haloalkoxy, hydroxy and amino;-   R¹³ is H or —(C₁-C₆alkylene)-T-R²¹; or-   R¹² and R¹³ can join to form a C₂-C₄alkylene group between the    oxygen atoms to which they are attached, wherein said C₂-C₄alkylene    group is optionally substituted with one C₆-C₁₀aryl group;-   R¹⁴ is H or C₁-C₆alkyl, phenyl, naphthyl or a 5 to 12 membered mono    or bicyclic heteroaryl containing 1, 2 or 3 heteroatoms    independently selected from N, O and S, which phenyl, naphthyl or    heteroaryl is optionally substituted with 1, 2 or 3 R²²;-   R¹⁵ and R^(15′) are each independently selected from H, C₁-C₆alkyl,    C₃-C₇cycloalkyl, C₃-C₇cycloalkylC₁-C₃alkyl, phenyl and benzyl, or    R¹⁵ and R^(15′) together with the carbon atom to which they are    attached from a C₃-C₇cycloalkylene group, wherein each C₁-C₆alkyl is    optionally substituted with a group selected from halo, OR¹⁸ and    SR¹⁸, and each C₃-C₇cycloalkyl, C₃-C₇cycloalkylene, phenyl and    benzyl is optionally substituted with one or two groups    independently selected from C₁-C₃alkyl, halo and OR¹⁸; or-   R^(15′) is H and R¹⁵ and R²⁴ together with the atoms to which they    are attached, form a 5-membered ring;-   R¹⁶ is H, C₁-C₆alkyl, C₂-C₁₀alkenyl, C₃-C₇cycloalkyl,    C₃-C₇cycloalkylC₁-C₃alkyl, benzyl, phenyl or adamantyl, any of which    is optionally substituted with 1, 2 or 3 groups, each independently    selected from halo, OR¹⁸ and N(R¹⁸)₂;-   each R¹⁷ is independently selected from H, C₁-C₆alkyl, C₂-C₆alkenyl,    C₂-C₆alkynyl, C₁-C₆haloalkyl, C₃-C₇cycloalkyl, C₃-C₇cycloalkenyl,    phenyl and benzyl; or-   both R¹⁷ together with the nitrogen atom to which they are attached    form a 3-7 membered heterocyclic or a 5-6 membered heteroaryl ring    which rings are optionally substituted with one or two groups    independently selected from C₁-C₃alkyl, halo, C₁-C₃haloalkyl, amino,    C₁-C₃alkylamino, (C₁-C₃alkyl)₂amino;-   each R¹⁸ is independently H, C₁-C₆alkyl, C₁-C₆haloalkyl or    C₃-C₇cycloalkyl;-   R¹⁹ is H, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl,    C₁-C₆haloalkyl, C₁-C₆hydroxyalkyl, halo, —OR¹⁸ or N(R¹⁸)₂;-   each R²⁰ is independently H, C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₇cycloalkyl, C₁-C₆hydroxyalkyl or C₃-C₇cycloalkylC₁-C₃alkyl;-   each R²¹ is independently H, C₁-C₆alkyl, C₁-C₆haloalkyl,    C₁-C₆hydroxyalkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl or    C₃-C₇cycloalkenyl;-   each R²² is independently selected from halo, C₁-C₆alkyl,    C₂-C₆alkenyl, C₁-C₆haloalkyl, phenyl, hydroxyC_(l)-C₆alkyl,    C₃-C₆cycloalkyl, C₁-C₆alkylcarbonyl, C₃-C₆cycloalkylcarbonyl,    carboxyC₁-C₆alkyl, oxo (required to make flavone), OR²⁰, SR²⁰,    N(R²⁰)₂, CN, NO₂, C(O)OR²⁰, C(O)N(R²⁰)₂ and NHC(O)R²⁰, or any two    R²² groups attached to adjacent ring carbon atoms can combine to    form —O—R²³—O—;-   R²³ is —[C(R³³)₂]_(n)-;-   R²⁴ is H, or R²⁴ and R¹⁵ together with the atoms to which they are    attached, form a 5-membered ring;-   each R³⁰ is independently selected from C₁-C₆alkyl and C₁-C₆alkoxy;-   each R³¹ is independently selected from H, C₁-C₆alkyl,    C₃-C₇cycloalkyl and benzyl;-   each R³² and R^(32′) is independently selected from H and    C₁-C₃alkyl;-   each R³³ is independently selected from H and C₁-C₆alkyl;-   U is O or S;-   each T is independently —S—, —O—, —SC(O)—, —C(O)S—, —SC(S)—,    —C(S)S—, —OC(O)—, —C(O)O— and —OC(O)O—;-   or a pharmaceutically acceptable salt and/or solvate thereof.

The compounds of formula I may optionally be provided in the form of apharmaceutically acceptable salt and/or solvate. In one embodiment thecompound of the invention is provided in the form of a pharmaceuticallyacceptable salt. In a second embodiment the compound of the invention isprovided in the form of a pharmaceutically acceptable solvate. In athird embodiment the compound of the invention is provided in its freeform.

In one aspect, the invention includes prodrugs. In a typicalconfiguration, the prodrug group is located at the 3′- and/or the5′-position of the sugar moiety. Suitable groups for this purposeinclude esters, i.e. groups of the formula OC(═O)R³⁰ wherein R³⁰typically is C₁-C₄alkyl, and amino acid esters, i.e. groups of theformula OC(═O)CHR³¹NH₂ wherein R³¹ typically is C₁-C₆alkyl. Furthersuitable prodrug groups are phosphate prodrugs, i.e. a prodrug groupwhich in vivo is converted to a phosphate. Prodrug group(s) may also bepresent on the nucleobase B.

In one embodiment of the invention, B is the group (a). Typically inthis embodiment, the group B is of the formula (a′):

wherein R⁵ is H or F, and R⁶ is N(R¹⁸)₂ or NHCOC₁-C₆alkyl. Typically R⁶is NH₂ .

In a further typical embodiment of the invention, B is of the group(a″):

wherein R⁶ is N(R¹⁸)₂ or NHCOC₁-C₆alkyl. Typically R⁶ is NH₂.

In a second embodiment of the invention, B is the group (b) Typically inthis embodiment, the group B is of the formula b′:

wherein R⁸ is H or F. Typically R⁸ is H

In a third embodiment of the invention B is the group (c′).

wherein R⁹ is OH or C₁-C₆alkoxy, and R¹⁰ is NH₂ or NHCOC₁-C₆alkyl.

In a fourth embodiment of the invention B is the group (d).

In one embodiment of the invention, R² is H.

In alternative embodiments of the invention, R² is C(═O)R³⁰,C(═O)CHR³¹NH₂ or OCR³²R^(32′)OC(═O)CHR³³NH₂.

In embodiments of the invention where R² is C(═O)R³⁰, R³⁰ is typicallymethyl, isopropyl, isobutyl or sec-butyl, especially isopropyl. Inembodiments of the invention where R² is C(═O)CHR³¹NH₂, R³¹ suitablycorresponds to the side chain of a natural or non-natural amino acid,such as the side chain of glycine (Gly), alanine (Ala), valine (Val),isoleucine (Ile) or phenylalanine (Phe), i.e. R³¹ is H, methyl,isopropyl, isobutyl or benzyl respectively, especially isopropyl. Ofparticular interest are amino acid ester moieties wherein theconfiguration at the asymmetric carbon atom to which R³¹ is attached isthat of an L-amino acid, in particular L-Ala, L-Val, L-Ile, and L-Phe,especially L-valine, i.e. R³¹ is isopropyl. In embodiments of theinvention where R² is OCR³²R^(32′)OC(═O)CHR³³NH₂, R³² and R^(32′) may bethe same or different and are typically selected from H and methyl, withR³³ typically being C₁-C₃alkyl.

In one embodiment of the invention, R¹ is H.

In alternative embodiments of the invention R¹ is a prodrug moiety.Suitably according to these embodiments R¹ is C(═O)R³⁰, C(═O)CHR³¹NH₂ orOCR³²R^(32′)OC(═O)CHR³³NH₂.

In embodiments of the invention where R¹ is C(═O)R³⁰, R³⁰ is typicallymethyl, isopropyl, isobutyl or sec-butyl, especially isopropyl. Inembodiments of the invention where R¹ is C(═O)CHR³¹NH₂, R³¹ suitablycorresponds to the side chain of a natural or non-natural amino acid,such as the side chains of glycine (Gly), alanine (Ala), valine (Val),isoleucine (Ile) or phenylalanine (Phe), i.e. R³¹ is H, methyl,isopropyl, isobutyl or benzyl respectively, especially isopropyl. Ofparticular interest are amino acid ester moieties wherein theconfiguration at the asymmetric carbon atom to which R³¹ is attached isthat of an L-amino acid, in particular L-Ala, L-Val, L-Ile, and L-Phe,especially L-valine, i.e. R³¹ is isopropyl. R³¹ may also be sec-butyl.In embodiments of the invention where R¹ is OCR³²R^(32′)OC(═O)CHR³³NH₂,R³² and R^(32′) may be the same or different and are typically selectedfrom H and methyl, with R³³ typically being H or C₁-C₃alkyl.

In one embodiment of the invention, R¹ and R² form together a bivalentlinker of the formula:

wherein R³ is as defined above, thus providing compounds of the formula:

Typically according to this embodiment, U is O.

Representative configurations for R³ include C₁-C₆alkoxy andNHC(R¹⁵)(R^(15′))C(═O)R¹⁶.

Typically, R³ is C₁-C₃alkoxy, such as isopropoxy or methoxy.

A further typical configuration for R³ is NHC(R¹⁵)(R^(15′))C(═O)R¹⁶.

Typically in this configuration, R¹⁵ and R^(15′) are each independentlyselected from H, C₁-C₆alkyl and benzyl. Typically, one of R¹⁵ andR^(15′) is H and the other is the side chain of an amino acid, such asthe side chain of alanine, valine, leucine or isoleucine, i.e. methyl,isopropyl, isobutyl or 1-methylprop-1-yl respectively. In a preferredconfiguration, one of R¹⁵ and R^(15′) is H and the other is methyl.

R¹⁶ is typically straight or branched C₁-C₆alkyl or C₃-C₇cycloalkyl. R¹⁶is isopropyl.

A representative value for R³ is NHCH(C₁-C₆alkyl)C(═O)C₁-C₃alkyl.

An alternative configuration for R³ is O—(C₁-C₆alkylene)-T-R²¹, whereinthe C₁-C₆alkylene moiety is linear or branched.

In one embodiment of compounds of formula (I), R¹ is the group (i):

Preferably in compounds according to this embodiment, U is O.

In one configuration of the group (i), R¹³ is H and R¹² is(C₁-C₆alkylene)-T-R²¹, typically in this configuration, R¹² is ethylene,T is O and R²¹ is C₁₂-C₁₉, thus forming the structure (i-a):

wherein n is an integer from 11 to 23, such as from 11 to 18. Preferablyn is an integer from 15 to 16.

Typically in the group (i-a), U is O.

Typically in compounds of formula (I) wherein R¹ is the group (i-a), R²is H.

In an alternative configuration of the group (i), R¹² and R¹³ join toform an optionally substituted C₂-C₄alkylene group between the oxygenatoms to which they are attached, thus forming a cyclic phosphate.Typically, the alkylene group is a C₃alkylene, thus providing thestructure (i-b):

Typically U is O and Ar is phenyl which is optionally substituted withone or two substituents independently selected from halo, C₁-C₆alkyl,C₁-C₆alkoxy and cyano, typically halo. Representative examples of Arinclude phenyl and phenyl which is substituted with chloro in the metaposition.

Typically in compounds of formula (I) wherein R¹ is the group (i-h), R²is H.

In a further configuration of the group (i), R¹³ is(C₁-C₆alkylene)-T-R²¹, thus providing the group (i-c):

wherein the C₁-C₆alkylene moiety is linear or branched. Non-limitingexamples of the C₁-C₆alkylene moiety in the group (i-c) includemethylene, ethylene, isopropylene and dimethylmethylene.

Typically in the group (i-c) U is O.

In a typical subgroup of the group (i-c), U is O, C₁-C₆alkylene ismethylene and T is —C(O)O—, or the C₁-C₆alkylene is ethylene and T is—C(O)S— thus providing compounds of formula I having any one of thepartial structures (i-c1) or (i-c2) respectively:

wherein R²¹ is C₁-C₆alkyl, such as tert-butyl. R¹² in these structuresis typically the same group as R¹³, or alternatively, R¹² is as definedabove.

Typically in compounds of formula (I) wherein R¹ is the group (i-c), R²is H.

In a further embodiment of compounds of formula (I), R¹ is the group(iii), i.e. R¹ together with the oxygen atom to which is attached, forma triphosphate, or a tri-thiophosphate, thus providing compounds havingthe structure:

or a pharmaceutically acceptable salt thereof, such as the potassiumsalt or the sodium salt. In preferred configurations according to theseembodiments, U is O.

Typically according to this embodiment, R² is H.

In a preferred embodiment, the invention provides a compound of theformula:

In a further embodiment of compounds of formula (I). R¹ is the group(iv):

In typical compounds of formula (I) wherein R¹ is the group (iv) and oneof R¹⁵ and R^(15′) is H, the stereochemistry is as indicated in thepartial formula:

U is typically O.

R²⁴ is typically H.

Representative examples of R¹⁴ include phenyl which is optionallysubstituted with one or two R²², wherein each R²² is independentlyselected from halo, C₁-C₆alkyl, C₂-C₆alkenyl and OR²⁰ and R²⁰ isC₁-C₆alkyl; or R¹⁴ is naphthyl.

Typically according to this embodiment, U is O, R²⁴ is H and R¹⁴ isphenyl which is optionally substituted with 1, 2 or 3 R²², thusproviding the group (iv-a):

In a typical configuration of the group (iv-a), the phenyl issubstituted with one or two halo, such as chloro or fluoro.

In a further representative configuration of group (iv-a), the phenyl issubstituted with one R²² which is selected from C₃-C₆cycloalkyl,C₁-C₆alkylcarbonyl or C₃-C₆cycloalkylcarbonyl, the cycloalkyl moietybeing optionally substituted with C₁-C₃alkyl.

In a further representative configuration of group (iv-a), the phenyl issubstituted with two R²², whereof one R²² is selected fromC₃-C₆cycloalkyl, C₁-C₆alkylcarbonyl or C₃-C₆cycloalkylcarbonyl, thecycloalkyl moiety being optionally substituted with C₁-C₃alkyl, and theother R²² is methyl, cyclopropyl, fluoro or chloro.

A further representative value for R¹⁴ is phenyl which is substitutedwith two R²² located on adjacent carbon atoms and the two R²² combine toform —O—CH₂—O—, thus forming the partial structure:

A further representative configuration of R¹⁴ is phenyl which issubstituted with R²² and R²² is carboxyC₁-C₆alkyl, and R²⁴ is H. Arepresentative example of this configuration is illustrated in formula(iv-b)

Typically in the group (iv-b), U is O.

In one configuration of the group (iv), R¹⁴ is phenyl which is fused toa 4-membered heterocyclic ring, which ring is substituted with keto andphenyl. Typical such structures are as shown in the partial formulaebelow:

Further representative values for R¹⁴ include indolyl, typically5-indolyl.

In one embodiment, R¹⁴ is heteroaryl, which heteroaryl is a 5 to 12membered mono or bicyclic aromatic ring containing 1, 2 or 3 heteroatomsindependently selected from N, O and S, and which heteroaryl isoptionally substituted with 1, 2 or 3 R²². Typically in this embodiment,each R²² is independently selected from C₁-C₆alkyl, C₂-C₆alkenyl,C₁-C₆haloalkyl, C₁-C₆alkoxy, hydroxy and amino.

A representative value for R¹⁴ according to this embodiment isoptionally substituted pyridyl.

Typical compounds according to this embodiment are those wherein U is Oand R¹⁴ is pyridyl which is optionally substituted with one or twosubstituents each independently selected from halo, C₁-C₆haloalkyl,C₁-C₆alkyl, C₂-C₆alkenyl, C₁-C₆alkoxy, hydroxy, amino.

Typically in compounds of formula (I) wherein R¹ is the group (iv), orany subgroup thereof, the moiety N(R²⁴)C(R¹⁵)(R^(15′))—C(═O)OR¹⁶ formsan amino acid ester residue, including natural and non-natural aminoacid residues. Typically one of R¹⁵ and R^(15′) is hydrogen, and theother one is hydrogen or C₁-C₆alkyl, such as isopropyl or isobutyl. Ofparticular interest are amino acid residues wherein R^(15′)is hydrogen,examples are glycine, (Gly) alanine (Ala), valine (Val), isoleucine(Ile) and phenylalanine (Phe) residues, i.e., R^(15′)is H and R¹⁵ ismethyl, isopropyl, isobutyl or benzyl respectively. In compounds whereinR^(15′) is hydrogen and R¹⁵ is other than hydrogen, the configuration atthe asymmetric carbon atom is typically that of an L-amino acid, inparticular L-Ala, L-Val, L-Ile, and L-Phe.

In a typical configuration of the group (iv), one of R¹⁵ and R^(15′) isH and the other is methyl.

In a further configuration of the group (iv), R¹⁵ and R^(15′) togetherwith the carbon atom to which they are attached form C₃-C₇cycloalkyl,for example cyclopropyl or cyclobutyl.

In a typical configuration of group (iv) R¹⁶ is C₁-C₁₀alkyl.

In one configuration of group (iv), R¹⁶ is C₁-C₃alkyl, such as methyl,ethyl, propyl, isopropyl, preferably isopropyl.

In a further configuration of group (iv), R¹⁶ is C₁-C₈alkyl, such as2-ethylbutyl, 2-pentyl, 2-butyl, isobutyl, tert-pentyl, preferably2-ethylbutyl.

In a further configuration of group (iv), R¹⁶ is C₃-C₇cycloalkyl, suchas cyclohexyl.

In one embodiment of compounds of formula (I), R¹ is the group (iv)wherein

-   U is O-   R²⁴ is H,-   R¹⁴ is phenyl which is substituted with C₃-C₆cycloalkyl,    C₁-C₆alkylcarbonyl or a 5- or 6-membered heteroaryl,-   R¹⁵ is H, R^(15′) is C₁-C₃alkyl, such as methyl, ethyl or isopropyl,    and-   R¹⁶ is C₁-C₆alkyl or C₃-C₇cycloalkyl, such as cyclopropyl,    cyclobutyl or cyclopentyl.

In one embodiment of compounds of formula (I). R¹ is the group (iv)wherein

-   R²⁴ is H,-   R¹⁴ is optionally substituted phenyl or naphthyl;-   R¹⁵ and R^(15′) are each independently H or C₁-C₆alkyl, and-   R¹⁶ is C₁-C₈alkyl or C₃-C₇cycloalkyl.

In a typical configuration of R¹ according to this embodiment

-   R²⁴ is H,-   R¹⁴ is optionally substituted phenyl;-   One of R¹⁵ and R^(15′) is H, and the other one is C₁-C₃alkyl, and-   R¹⁶ is C₁-C₈alkyl.

In a typical embodiment the invention includes compounds wherein

-   B is the group b′;-   U is O;-   R¹ is the group (iv);-   R² is H;-   R⁸ is H;-   R¹⁴ is optionally substituted phenyl;-   one of R¹⁵ and R^(15′) is H is and the other is C₁-C₃alkyl;-   R¹⁶ is C₁-C₈alkyl.

In a preferred embodiment, the invention provides a compound of theformula:

In a further preferred embodiment, the invention provides a compound ofthe formula:

the compound being a single stereoisomer at the phosphorus atom.

In a further preferred embodiment, the invention provides a compound ofthe formula:

the compound being a single stereoisomer at the phosphorus atom.

In a further preferred embodiment, the invention provides a compound ofthe formula:

In an alternative configuration of the group (iv), R¹⁵ is H, and R^(15′)and R²⁴ together with the atoms to which they are attached form apyrrolidine ring, thus affording the group (iv-c):

Typically in this configuration, U is O, R¹⁴ is optionally substitutedphenyl and R¹⁶ is C₁-C₆alkyl or C₃-C₆cycloalkyl.

Typically in compounds of formula (I) wherein R¹ is the group (iv), orany subgroup thereof, R² is H.

In a further embodiment of compounds of formula (I), R¹ is the group(v):

Typically in the group (v), U is O.

According to this embodiment, the two N-linked substituents to theP-atom are identical, i.e. both of the R¹⁵ moieties are the same, bothof the R^(15′) moieties are the same, and both of R¹⁶ moieties are thesame.

In a typical configuration of the group (v) both R¹⁵ are H or C₁-C₆alkyl(such as ethyl, n-propyl, isopropyl, n-butyl or isobutyl), both R^(15′)are H, and both R¹⁶ are C₁-C₆alkyl (such as methyl, ethyl or isopropyl)or C₃-C₇cycloalkyl (such as cyclopropyl, cyclobutyl or cyclopentyl).

In one configuration of group (v), R¹⁶ is C₁-C₃alkyl, such as methyl,propyl, isopropyl, preferably isopropyl.

In a further configuration of group (v), R¹⁶ is C₁-C₈alkyl, such as2-ethylbutyl, 2-pentyl, 2-butyl, isobutyl, tert-pentyl, preferably2-ethylbutyl.

In a further configuration of group (v), R¹⁶ is C₃-C₇cycloalkyl, such ascyclohexyl

In a further embodiment of compounds of formula (I), R¹ is the group(vi):

Typically in the group (vi), U is O.

In one configuration of the group (vi), R¹³ is —(C₁-C₆alkylene)-T-R²¹,thus providing the structure (vi-a):

wherein the C₁-C₆alkylene moiety is linear or branched. Non-limitingexamples of the C₁-C₆alkylene moiety in the group (vi-a) includemethylene, ethylene, isopropylene and dimethylmethylene.

In one configuration of the subgroup vi-a, R²¹ is1-hydroxy-2-methylpropan-2-yl, i.e. a group of the formula:

Typically in the group (vi-a), U is O.

In a typical subgroup of the group (vi-a), C₁-C₆alkylene is methylenewhich is optionally substituted with one or two C₁-C₃alkyl, and T is—OC(O)O—, thus providing compounds of formula I having of the partialstructure (vi-b):

wherein R³² and R^(32′) are independently H or C₁-C₃alkyl. Typically,one of R³² and R^(32′) is H and the other one is H, methyl or isopropyl.Alternatively, R³² and R^(32′) are both methyl.

Typically in the group (vi-b), U is O.

Typical examples of R²¹ include optionally substituted C₁-C₆alkyl, suchas methyl, ethyl, propyl and isopropyl.

Typically, one of R¹⁷ and R^(17′) is H and the other one is phenyl orbenzyl, preferably benzyl.

Typically in compounds of formula (I) wherein R¹ is the group (vi) orany subgroup thereof, R² is H.

In a further subgroup of the group (vi-a), U is O, C₁-C₆alkylene isethylene and T is —C(O)S—, thus providing compounds of formula I havingof the partial structure:

Typical examples of R²¹ include optionally substituted. C₁-C₆alkyl,especially branched C₁-C₆alkyl, and C₁-C₆hydroxyalkyl.

Typically, one of R¹⁷ and R^(17′) is H and the other one is phenyl orbenzyl, preferably benzyl.

Typically in compounds of formula (I) wherein R¹ is the group (vi) orany subgroup thereof, R² is H.

In another aspect, the present invention provides compounds representedby formula:

wherein:

-   B is a nucleobase selected from the groups (a) to (d):

-   -   wherein Y is N or C(R¹⁹);

-   R¹ is the group (v):

-   R⁴, R⁵, R⁷ and R⁸ are each independently H, C₁-C₆alkyl,    C₁-C₆haloalkyl, C₁-C₆hydroxyalkyl, halo, —OR¹⁸, —SR¹⁸ or —N(R¹⁸)₂;-   R⁶, R⁹, R¹⁰, R¹¹ are each independently selected from H, C₁-C₆alkyl,    C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl, C₁-C₆haloalkyl,    C₁-C₆hydroxyalkyl, halo, OR¹⁸, SR¹⁸, N(R¹⁸)₂, NHC(O)OR¹⁸,    NHC(O)N(R¹⁸)₂, CN, NO₂, C(O)R¹⁸, C(O)OR¹⁸, C(O)N(R¹⁸)₂ and    NHC(O)R¹⁸, wherein said C₂-C₆alkenyl group and said C₂-C₆alkynyl    group can be optionally substituted with halo or C₃-C₅cycloalkyl;-   R¹⁵ and R^(15′) are each independently selected from H, C₁-C₆alkyl,    C₃-C₇cycloalkyl, C₃-C₇cycloalkylC₁-C₃alkyl, phenyl and benzyl, or    R¹⁵ and R^(15′) together with the carbon atom to which they are    attached from a C₃-C₇cycloalkylene group, wherein each C₁-C₆alkyl is    optionally substituted with a group selected from halo, OR¹⁸ and    SR¹⁸, and each C₃-C₇cycloalkyl, C₃-C₇cycloalkylene, phenyl and    benzyl is optionally substituted with one or two groups    independently selected from C₁-C₃alkyl, halo and OR¹⁸; or-   R^(15′) is and R¹⁵ and R²⁴ together with the atoms to which they are    attached, form a 5-membered ring;-   R¹⁶ is H, C₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₃-C₇cycloalkyl,    C₃-C₇cycloalkylC₁-C₃alkyl, benzyl, phenyl or adamantyl, any of which    is optionally substituted with 1, 2 or 3 groups, each independently    selected from halo, OR¹⁸ and N(R¹⁸)₂;-   each R¹⁸ is independently H, C₁-C₆alkyl, C₁-C₆haloalkyl or    C₃-C₇cycloalkyl;-   R¹⁹ is H, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl,    C₁-C₆haloalkyl, C₁-C₆hydroxyalkyl, halo, —OR¹⁸ or N(R¹⁸)₂;-   each R²⁰ is independently H, C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₇cycloalkyl, C₁-C₆hydroxyalkyl or C₃-C₇cycloalkylC₁-C₃alkyl;-   each R³⁰ is independently selected from C₁-C₆alkyl and C₁-C₆alkoxy;-   each R³¹ is independently selected from H, C₁-C₆ alkyl,    C₃-C₇cycloalkyl and benzyl;-   each R³² and R^(32′) is independently selected from H and    C₁-C₃alkyl;-   each R³³ is independently selected from H and C₁-C₆alkyl;-   U is O;-   or a pharmaceutically acceptable salt and/or solvate thereof.

In one embodiment of this aspect, the group B is (a′)

-   wherein-   R⁵ is H or F, and R⁶ is N(R¹⁸)₂ or NHCOC₁-C₆alkyl. Typically R⁶ is    NH₂.

In one embodiment of this aspect, the group B is (b′)

-   wherein R⁸ is H or F. Typically, R⁸ is H.

In one embodiment of this aspect, the group B is (c′)

-   wherein R⁹ is OH or C₁-C₆alkoxy, and R¹⁰ is NH₂ or NHCOC₁-C₆alkyl.

Typically in this aspect, R² is H.

In another aspect, the present invention provides compounds representedby formula VI:

wherein:

-   B is a nucleobase selected from the groups (a) to (d):

wherein Y is N or C(R¹⁹);

-   R¹ is the group

-   R² is H, C(═O)R³⁰, C(═O)CHR³¹NH₂, CR³²R^(32′)OC(═O)CHR³³NH₂ or    CR³²R^(32′)OC(═O)³⁰;-   R⁴, R⁵, R⁷ and R⁸ are each independently H, C₁-C₆alkyl,    C₁-C₆haloalkyl, C₁-C₆hydroxyalkyl, halo, OR¹⁸, SR¹⁸ or N(R¹⁸)₂;-   R⁶, R⁹, R¹⁰, R¹¹ are each independently selected from H, C₁-C₆alkyl,    C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl, C₁-C₆haloalkyl,    C₁-C₆hydroxyalkyl, halo, OR¹⁸, SR¹⁸, N(R¹⁸)₂, NHC(O)OR¹⁸,    NHC(O)N(R¹⁸)₂, CN, NO₂, C(O)R¹⁸, C(O)OR¹⁸, C(O)N(R¹⁸)₂ and    NHC(O)R¹⁸, wherein said C₂-C₆alkenyl group and said C₂-C₆alkynyl    group can be optionally substituted with halo or C₃-C₅cycloalkyl;-   R¹³ is C₁-C₆alkylene-T-R²¹;-   each R¹⁸ is independently H, C₁-C₆alkyl, C₁-C₆haloalkyl or    C₃-C₇cycloalkyl;-   R¹⁹ is H, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl,    C₁-C₆haloalkyl, C₁-C₆hydroxyalkyl, halo, OR¹⁸ or N(R¹⁸)₂;-   each R²⁰ is independently H, C₁-C₆alkyl, C₁-C₆haloalkyl,    C₃-C₇cycloalkyl, C₁-C₆hydroxyalkyl or C₃-C₇cycloalkylC₁-C₃alkyl;-   each R²¹ is independently H, C₁-C₂₄haloalkyl, C₁-C₆haloalkyl,    C₁-C₆hydroxyalkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl or    C₃-C₇cycloalkenyl;-   each R³⁰ is independently selected from C₁-C₆alkyl and C₁-C₆alkoxy;-   each R³¹ is independently selected from H, C₁-C₆alkyl,    C₃-C₇cycloalkyl and benzyl;-   each R³² and R^(32′) is independently selected from H and    C₁-C₃alkyl;-   each R³³ is independently selected from H and C₁-C₆alkyl;-   U is O;-   each T is independently S, O, SC(O), C(O)S, SC(S), C(S)S, OC(O),    C(O)O and OC(O)O;-   or a pharmaceutically acceptable salt and/or solvate thereof.

In one embodiment of this aspect, the group B is (a′)

-   wherein-   R⁵ is H or F, and R⁶ is N(R¹⁸)₂ or NHCOC₁-C₆alkyl. Typically R⁶ is    NH₂.

In one embodiment of this aspect, the group B is (b′)

wherein R⁸ is H or F. Typically, R⁸ is H.

In one embodiment of this aspect, the group B is (c′)

wherein R⁹ is OH or C₁-C₆alkoxy, and R¹⁰ is NH₂ or NHCOC₁-C₆alkyl.

Typically in this aspect, R² is H.

Consequently, there is provided a compound of formula I for use as amedicament, in particular for use in the treatment or prophylaxis of HCVinfection, especially the treatment of HCV infection.

Further provided is the use of a compound of formula I in themanufacture of a medicament, in particular a medicament for thetreatment or prophylaxis of HCV infection, especially a medicament forthe treatment of HCV infection.

Additionally, there is provided a method for the treatment orprophylaxis of HCV infection comprising, the administration of acompound of formula I, in particular a method for the treatment of HCVinfection comprising the administration of a compound of formula I.

In a further aspect, the invention concerns the use of the compounds ofthe invention for inhibiting HCV.

Additionally, there is provided the use of the compounds of formula Ifor the treatment or prophylaxis of HCV infection, such as the treatmentor prophylaxis of HCV infection in humans.

In a preferred aspect, the invention provides the use of compounds offormula I for the treatment of HCV infection, such as the treatment ofHCV infection in humans.

Furthermore, the invention relates to a method for manufacturingcompounds of formula I, to novel intermediates of use in the manufactureof compounds of formula I and to the manufacture of such intermediates.

In a further aspect, the invention provides pharmaceutical compositionscomprising a compound of formula I in association with apharmaceutically acceptable adjuvant, diluent, excipient or carrier. Thepharmaceutical composition will typically contain an antivirallyeffective amount (e.g. for humans) of the compound of formula I,although sub-therapeutic amounts of the compound of formula I maynevertheless be of value when intended for use in combination with otheragents or in multiple doses.

The skilled person will recognise that references to compounds offormula I will include any subgroup of the compounds of formula Idescribed herein.

HCV genotypes in the context of treatment or prophylaxis in accordancewith the invention include the major HCV genotypes, i.e. genotype 1a,1b, 2a, 3a, 4a, 5a and 6a. The invention also provides a method for thetreatment or prophylaxis of HCV infection. Typically, the inventionprovides a method for the treatment of HCV infection.

Representative HCV genotypes in the context of treatment or prophylaxisin accordance with the invention include genotype 1b (prevalent inEurope) and 1a (prevalent in North America). The invention also providesa method for the treatment or prophylaxis of HCV infection, inparticular of the genotype 1a or 1b. Typically, the invention provides amethod for the treatment of HCV infection, in particular of the genotype1a or 1b.

Further representative genotypes in the context of treatment orprophylaxis in accordance with the invention include genotype 3a, suchas wild type genotype 3a and mutant strains of the genotype 3a, forexample the S282T and L159/320F mutants. Typically, the inventionprovides a method for the treatment of HCV infection, in particular ofthe genotype 3a, such as wild type genotype 3a and mutant strains of thegenotype 3a, for example the S282T and L159/320F mutants.

The invention further relates to the treatment or prophylaxis of HCVinfection caused by the genotypes 2a, 4a, 5a, 6a. The invention alsoprovides a method for the treatment or prophylaxis of HCV infection, ofthe genotypes 2a, 4a, 5a, 6a.

The good activity of the compounds of the invention against genotype 3is noteworthy given the poor performance of previous generations ofnucleotides. Preferably the compositions of the invention havepan-genotypic coverage against each of the 6 genotypes, that is the EC₅₀of the compound of the invention does not differ markedly betweengenotypes, thereby simplifying treatment.

The compounds of the invention have several chiral centers and may existand be isolated in optically active and racemic forms. Some compoundsmay exhibit polymorphism. It is to be understood that any racemic,optically active, diastereomeric, polymorphic or stereoisomeric form ormixtures thereof, of a compound provided herein is within the scope ofthis invention. The absolute configuration of such compounds can bedetermined using methods known in the art such as, for example, X-raydiffraction or NMR and/or implication from starting materials of knownstereochemistry and/or stereoselective synthesis methods. Pharmaceuticalcompositions in accordance with the invention will preferably comprisesubstantially stereoisomerically pure preparations of the indicatedstereoisomer.

Most amino acids are chiral and can exist as separate enantiomers. Theyare designated L- or D-amino acids, wherein the L-enantiomer is thenaturally occurring enantiomer. Accordingly, pure enantiomers of theamino acids are readily available and where an amino acid is used in thesynthesis of a compound of the invention, the use of a chiral aminoacid, will provide a chiral product.

Pure stereoisomeric forms of the compounds and intermediates asmentioned herein are defined as isomers substantially free of otherenantiomeric or diastereomeric forms of the same basic molecularstructure of said compounds or intermediates. In particular, the term“stereoisomerically pure” concerns compounds or intermediates having astereoisomeric excess of at least 80% (i.e. minimum 90% of one isomerand maximum 10% of the other possible stereoisomers) up to astereoisomeric excess of 100% (i.e. 100% of one isomer and none of theother), more in particular, compounds or intermediates having astereoisomeric excess of 90% up to 100%, even more in particular havinga stereoisomeric excess of 94% up to 100% and most in particular havinga stereoisomeric excess of 97% up to 100%. The terms “enantiomericallypure” and “diastereomerically pure” should be understood in a similarway, but then having regard to the enantiomeric excess, and thediastereomeric excess, respectively, of the mixture in question.

Pure stereoisomeric forms of the compounds and intermediates of thisinvention may be obtained by using procedures well known in the art. Forinstance, enantiomers may be separated from each other by resolution ofthe racemic mixture, i.e. formation of a diastereomeric salt effected byreaction with an optically active acid or base followed by selectivecrystallization of the formed diastereomeric salt. Examples of suchacids are tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acidand camphorsulfonic acid. Alternatively, enantiomers may be separated bychromatographic techniques using chiral stationary phases. Purestereochemically isomeric forms may also be obtained by synthesis fromstereochemically pure forms of the appropriate starting materials,provided that the reaction occurs stereospecifically, by chiralsynthesis or by utilisation of a chiral auxiliary. If a specificstereoisomer is desired, the preparation of that compound is preferablyperformed using stereospecific methods. These methods willadvantageously employ enantiomerically pure starting materials.

Diastereomeric racemates of the compounds of the invention can beseparated by conventional methods. Appropriate physical separationmethods that may advantageously be employed are, for example, selectivecrystallization and chromatography, e.g. column chromatography.

When a phosphorus atom is present in compounds of the inventioncomprising, the phosphorus atom may represent a chiral centre. Thechirality at this centre is designated “R” or “S” according to theCahn-Angold-Prelog priority rules. When the chirality is not indicated,it is contemplated that both the R- and S-isomers are meant to beincluded, as well as a mixture of both.

In preferred embodiments of the invention compounds of formula I or anysubgroup of formula I which are pure stereoisomers at the phosphorusatom are included. Preferred are compounds having the S-configuration atthe phosphorus atom. These stereoisomers are designated S_(P).

In other embodiments of the invention, compounds of formula I or anysubgroup of formula I having the R-configuration at the phosphorus atomare included. These stereoisomers are designated R_(P).

In other embodiments of the invention, diastereomeric mixtures areincluded, i.e. mixtures of compounds having the R— or S— configurationat the phosphorus atom.

The present invention also includes isotope-labelled compounds offormula I or any subgroup of formula I, wherein one or more of the atomsis replaced by an isotope of that atom, i.e. an atom having the sameatomic number as, but an atomic mass different from, the one(s)typically found in nature. Examples of isotopes that may be incorporatedinto the compounds of formula I or any subgroup of formula include butare not limited to isotopes of hydrogen, such as ²H and ³H (also denotedD for deuterium and T for tritium, respectively), carbon, such as ¹¹C,¹³C and ¹⁴C, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and¹⁸O, phosphorus, such as ³¹P and ³²P, sulfur, such as ³⁵S, fluorine,such as ¹⁸F, chlorine, such as ³⁶Cl, bromine such as ⁷⁵Br, ⁷⁶Br, ⁷⁷Brand ⁸²Br, and iodine, such as ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I. The choice ofisotope included in an isotope-labelled compound will depend on thespecific application of that compound. For example, for drug orsubstrate tissue distribution assays, compounds wherein a radioactiveisotope such as ³H or ¹⁴C is incorporated will generally be most useful.For radio-imaging applications, for example positron emission tomography(PET) a positron emitting isotope such as ¹¹C, ¹⁸F, ¹³N or ¹⁵O will beuseful. The incorporation of a heavier isotope, such as deuterium, i.e.²H, may provide greater metabolic stability to a compound of formula Ior any subgroup of formula I, which may result in, for example, anincreased in vivo half life of the compound or reduced dosagerequirements.

Isotope-labelled compounds of formula I or any subgroup of formula I canbe prepared by processes analogous to those described in the Schemesand/or Examples herein below by using the appropriate isotope-labelledreagent or starting material instead of the correspondingnon-isotope-labelled reagent or starting material, or by conventionaltechniques known to those skilled in the art.

The pharmaceutically acceptable addition salts comprise thetherapeutically active non-toxic acid and base addition salt forms ofthe compounds of formula I. Of interest are the free, i.e. non-saltforms of the compounds of formula I.

The pharmaceutically acceptable acid addition salts can conveniently beobtained by treating the base form with such appropriate acid.Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propionic, hydroxyacetic, lactic pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic hydroxybutanedioic acid), tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

The compounds of formula I containing an acidic proton may also beconverted into their non-toxic metal or amine addition salt forms bytreatment with appropriate organic and inorganic bases. Appropriate basesalt forms comprise, for example, the ammonium salts, the alkali andearth alkaline metal salts, e.g. the lithium, sodium, potassium,magnesium, calcium salts and the like, salts with organic bases, e.g.the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts withamino acids such as, for example, arginine, lysine and the like.

Some of the compounds of formula I may also exist in their tautomericform. For example, tautomeric forms of amide groups (—C(═O)—NH—) areiminoalcohols (—C(OH)═N—), which can become stabilized in rings witharomatic character. Such forms, although not explicitly indicated in thestructural formulae represented herein, are intended to be includedwithin the scope of the present invention.

The terms and expressions used herein throughout the abstract,specification and claims shall be interpreted as defined below unlessotherwise indicated. The meaning of each term is independent at eachoccurrence. These definitions apply regardless of whether a term is usedby itself or in combination with other terms, unless otherwiseindicated. A term or expression used herein which is not explicitlydefined, shall be interpreted as having its ordinary meaning used in theart. Chemical names, common names, and chemical structures may be usedinterchangeably to describe the same structure. If a chemical compoundis referred to using both a chemical structure and a chemical name andan ambiguity exists between the structure and the name, the structurepredominates.

“C_(m)-C_(n)alkyl” on its own or in composite expressions such asC_(m)-C_(n)haloalkyl C_(m)-C_(n)alkylcarbonyl, C_(m)-C_(n)alkylamine,etc. represents a straight or branched aliphatic hydrocarbon radicalhaving the number of carbon atoms designated, e.g. C₁-C₄alkyl means analkyl radical having from 1 to 4 carbon atoms. C₁-C₆alkyl has acorresponding meaning, including also all straight and branched chainisomers of pentyl and hexyl. Preferred alkyl radicals for use in thepresent invention are C₁-C₆alkyl, including methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-buty, tert-butyl, n-pentyl andn-hexyl, especially C₁-C₄alkyl such as methyl, ethyl, n-propyl,isopropyl, t-butyl, n-butyl and isobutyl. Methyl and isopropyl aretypically preferred. An alkyl group may be unsubstituted or substitutedby one or more substituents which may be the same or different, eachsubstituent being independently selected from the group consisting ofhalo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, —O-alkyl,—O-aryl, -alkylene-O-alkyl, alkylthio, —NH₂, —NH(alkyl), —N(alkyl)₂,—NH(cycloalkyl), —O—C(═O)-alkyl, —O—C(═O)-aryl, —O—C(═O)-cycloalkyl,—C(═O)OH and —C(═O)O-alkyl. It is generally preferred that the alkylgroup is unsubstituted, unless otherwise indicated.

“C₂-C_(n)alkenyl” represents a straight or branched aliphatichydrocarbon radical containing at least one carbon-carbon double bondand having the number of carbon atoms designated, e.g. C₂-C₄alkenylmeans an alkenyl radical having from 2 to 4 carbon atoms; C₂-C₆alkenylmeans an alkenyl radical having from 2 to 6 carbon atoms. Non-limitingalkenyl groups include ethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl,n-pentenyl and hexenyl. An alkenyl group may be unsubstituted orsubstituted by one or more substituents which may be the same ordifferent, each substituent being independently selected from the groupconsisting of halo, alkenyl, alkynyl aryl, cycloalkyl, cyano, hydroxy,—O-alkyl, —O-aryl, -alkylene-O-alkyl, alkylthio, —NH₂, —NH(alkyl),—NH(alkyl)₂, —N(cycloalkyl), —O—C(═O)-alkyl, —O—C(═O))-aryl,—O—C(═O)-cycloalkyl, —C(═O)OH and —C(═O)O-alkyl. It is generallypreferred that the alkynyl group is unsubstituted, unless otherwiseindicated.

“C₂-C_(n)alkynyl” represents a straight or branched aliphatichydrocarbon radical containing at least one carbon-carbon tripple bondand having the number of carbon atoms designated, e.g. C₂-C₄alkynylmeans an alkynyl radical having from 2 to 4 carbon atoms; C₂-C₆alkynylmeans an alkynyl radical having from 2 to 6 carbon atoms. Non-limitingalkenyl groups include ethynyl, propynyl, 2-butynyl and 3-methylbutynylpentynyl and hexynyl. An alkynyl group may be unsubstituted orsubstituted by one or more substituents which may be the same ordifferent, each substituent being independently selected from the groupconsisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy,—O-alkyl, —O-aryl, -alkylene-O-alkyl, alkylthio, —NH₂, —NH(alkyl),—N(alkyl)₂, —NH(cycloalkyl), —O—C(O)-alkyl, —O—C(O)-aryl,—O—C(O)-cycloalkyl, —C(O)OH and —C(O)O-alkyl. It is generally preferredthat the alkynyl group is unsubstituted, unless otherwise indicated.

The term “C_(m)-C_(n)haloalkyl” as used herein representsC_(m)-C_(n)alkyl wherein at least one C atom is substituted with ahalogen (e.g., the C_(m)-C_(n)haloalkyl group may contain one to threehalogen atoms), preferably chloro or fluoro. Typical haloalkyl groupsare C₁-C₂haloalkyl, in which halo suitably represents fluoro. Exemplaryhaloalkyl groups include fluoromethyl, difluoromethyl andtrifluoromethyl.

The term “C_(m)-C_(n)hydroxyalkyl” as used herein representsC_(m)-C_(n)alkyl wherein at least one C atom is substituted with onehydroxy group. Typical C_(m)-C_(n)hydroxyalkyl groups areC_(m)-C_(n)alkyl wherein one C atom is substituted with one hydroxygroup. Exemplary hydroxyalkyl groups include hydroxymethyl andhydroxyethyl.

The term “C_(m)-C_(n)aminoalkyl” as used herein representsC_(m)-C_(n)alkyl wherein at least one C atom is substituted with oneamino group. Typical C_(m)-C_(n)aminoalkyl groups are C_(m)-C_(n)alkylwherein one C atom is substituted with one amino group. Exemplaryaminoalkyl groups include aminomethyl and aminoethyl.

The term “C_(m)-C_(n)alkylene” as used herein represents a straight orbranched bivalent alkyl radical having the number of carbon atomsindicated. Preferred C_(m)-C_(n)alkylene radicals for use in the presentinvention are C₁-C₃alkylene. Non-limiting examples of alkylene groupsinclude —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH(CH₃)CH₂CH₂—, —CH(CH₃)— and—CH(CH(CH₃)₂)—.

The term “Me” means methyl, and “MeO” means methoxy.

The term “C_(m)-C_(n)alkylcarbonyl” represents a radical of the formulaC_(m)-C_(n)alkyl-C(═O)— wherein the C_(m)-C_(n)alkyl moiety is asdefined above. Typically, “C_(m)-C_(n)alkylcarbonyl” isC₁-C₆alkyl-C(═O)—.

“C_(m)-C_(n)alkoxy” represents a radical C_(m)-C_(n)alkyl-O— whereinC_(m)-C_(n)alkyl is as defined above. Of particular interest isC₁-C₄alkoxy which includes methoxy, ethoxy, n-propoxy, isopropoxy,t-butoxy, n-butoxy and isobutoxy. Methoxy and isopropoxy are typicallypreferred. C₁-C₆alkoxy has a corresponding meaning, expanded to includeall straight and branched chain isomers of pentoxy and hexoxy.

The term “C_(m)-C_(n)alkoxycarbonyl” represents a radical of the formulaC_(m)-C_(n)alkoxy-C(═O)— wherein the C_(m)-C_(n)alkoxy moiety is asdefined above. Typically, “C_(m)-C_(n)alkoxycarbonyl” isC₁-C₆alkoxy-C(═O)—.

The term “amino” represents the radical —NH₂.

The term “halo” represents a halogen radical such as fluoro, chloro,bromo or iodo. Typically, halo groups are fluoro or chloro.

The term “aryl” means a phenyl biphenyl or naphthyl group.

The term “heterocycloalkyl” represents a stable saturated monocyclic 3-7membered ring containing 1-3 heteroatoms independently selected from O,S and N. In one embodiment the stable saturated monocyclic 3-7 memberedring contains 1 heteroatom selected from O, S and N. In a secondembodiment the stable saturated monocyclic 3-7 membered ring contains 2heteroatoms independently selected from O, S and N. In a thirdembodiment the stable saturated monocyclic 3-7 membered ring contains 3heteroatoms independently selected from O, S and N. The stable saturatedmonocyclic 3-7 membered ring containing 1-3 heteroatoms independentlyselected from O, S and N may typically be a 5-7 membered ring, such as 5or 6 membered ring. A heterocycloalkyl group may be unsubstituted orsubstituted by one or more substituents which may be the same ordifferent, each substituent being independently selected from the groupconsisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy,—O-alkyl, —O-aryl, -alkylene-O-alkyl, alkylthio, —NH₂, —NH(alkyl),—N(alkyl)₂, —NH(cycloalkyl), —O—C(O)-alkyl, —O—C(O)-aryl,—O—C(O)-cycloalkyl, —C(O)OH and —C(O)O-alkyl. It is generally preferredthat the heterocycloalkyl group is unsubstituted, unless otherwiseindicated.

The term “heteroaryl” represents a stable mono or bicyclic aromatic ringsystem containing 1-4 heteroatoms independently selected from O, S andN, each ring having 5 or 6 ring atoms. In one embodiment of theinvention the stable mono or bicyclic aromatic ring system contains oneheteroatom selected from O, S and N, each ring having 5 or 6 ring atoms.In a second embodiment of the invention the stable mono or bicyclicaromatic ring system contains two heteroatoms independently selectedfrom O, S and N, each ring having 5 or 6 ring atoms. In a thirdembodiment the stable mono or bicyclic aromatic ring system containsthree heteroatoms independently selected from O, S and N, each ringhaving 5 or 6 ring atoms. In a fourth embodiment the stable mono orbicyclic aromatic ring system contains four heteroatoms independentlyselected from O, S and N, each ring having 5 or 6 ring atoms.

One embodiment of heteroaryl comprises flavone.

The term “C₃-C_(n)cycloalkyl” represents a cyclic monovalent alkylradical having the number of carbon atoms indicated, e.g.C₃-C₇cycloalkyl means a cyclic monovalent alkyl radical having from 3 to7 carbon atoms. Preferred cycloalkyl radicals for use in the presentinvention are C₃-C₄alkyl i.e. cyclopropyl and cyclobutyl. A cycloalkylgroup may be unsubstituted or substituted by one or more substituentswhich may be the same or different, each substituent being independentlyselected from the group consisting of halo, alkenyl, alkynyl, aryl,cycloalkyl, cyano, hydroxy, —O-alkyl, —O-aryl, -alkylene-O-alkyl,alkylthio, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(cycloalkyl), —O—C(O)-aryl,—O—C(O)-cycloalkyl, —C(O)OH and —C(O)O-alkyl. It is generally preferredthat the cycloalkyl group is unsubstituted, unless otherwise indicated.

The term “aminoC_(m)-C_(n)alkyl” represents a C_(m)-C_(n)alkyl radicalas defined above which is substituted with an amino group, i.e. onehydrogen atom of the alkyl moiety is replaced by an NH₂-group.Typically, “aminoC_(m)-C_(n)alkyl” is aminoC₁-C₆alkyl.

The term “aminoC_(m)-C_(n)alkylcarbonyl” represents aC_(m)-C_(n)alkylcarbonyl radical as defined above, wherein one hydrogenatom of the alkyl moiety is replaced by an NH₂-group. Typically,“aminoC_(m)-C_(n)alkylcarbonyl” is aminoC₁-C₆alkylcarbonyl. Examples ofaminoC_(m)-C_(n)alkylcarbonyl include but are not limited to glycyl:C(═O)CH₂NH₂, alanyl: C(═O)CH(NH₂)CH₃, valinyl: C═OCH(NH₂)CH(CH₃)₂,leucinyl: C(═O)CH(NH₂)(CH₂)₃CH₃, isoleucinyl:C(═O)CH(NH₂)CH(CH₃)(CH₂CH₃) and norleucinyl: C(═O)CH(NH₂)(CH₂)₃CH₃ andthe like. This definition is not limited to naturally occurring aminoacids.

Related terms, are to be interpreted accordingly in line with thedefinitions provided above and the common usage in the technical field.

As used herein, the term “(═O)” forms a carbonyl moiety when attached toa carbon atom. It should be noted that an atom can only carry an oxogroup when the valency of that atom so permits.

The term “monophosphate, diphosphate and triphosphate ester” refers togroups:

The term “thio-monophosphate, thio-diphosphate and thio-triphosphateester” refers to groups:

As used herein, the radical positions on any molecular moiety used inthe definitions may be anywhere on such a moiety as long as it ischemically stable. When any variable present occurs more than once inany moiety, each definition is independent.

Whenever used herein, the term “compounds of formula I”, or “thecompounds of the invention” or similar terms, it is meant to include thecompounds of formula I and subgroups of compounds of formula I,including the possible stereochemically isomeric forms, and theirpharmaceutically acceptable salts and solvates.

The term “solvates” covers any pharmaceutically acceptable solvates thatthe compounds of formula I as well as the salts thereof, are able toform. Such solvates are for example hydrates, alcoholates, e.g.ethanolates, propanolates, and the like, especially hydrates.

In general, the names of compounds used in this application aregenerated using ChemDraw Ultra 12.0. In addition, if the stereochemistryof a structure or a portion of a structure is not indicated with forexample bold or dashed lines, the structure or portion of that structureis to be interpreted as encompassing stereoisomers of it.

General Synthetic Methods

Compounds of the present invention may be prepared by a variety ofmethods e.g. as depicted in the illustrative synthetic schemes shown anddescribed below. The starting materials and reagents used are availablefrom commercial suppliers or can be prepared according to literatureprocedures set forth in references using methods well known to thoseskilled in the art.

Scheme 1 illustrates a route to compounds of formula I wherein R¹ and R²are and the base B is uracil or derivatised uracil, i.e. B is a group offormula (b).

Protection of the hydroxy groups of(4S,5R)-4-hydroxy-5-(hydroxymethyl)dihydrofuran-2(3H)-one using forinstance triisopropylsilyl (TIPS) groups effected by treatment withTIPS-chloride in the presence of a base like imidazole or similar, orany other suitable protecting groups such as acyl groups like acetyl,benzoyl or p-chlorobenzoyl groups or trityl groups may be used.Alternatively, an orthogonal protecting group strategy may be employedin order to enable later selective deprotection of one of the hydroxygroups without touching the other. Typically then, the 5′-hydroxy groupis protected with a trityl, methoxytrityl or a silyl group, followed byprotection of the 3′-hydroxy group using e.g. an acyl group. The thusprotected derivative is then subjected to electrophilic α-fluorinationby treatment with N-fluorobenzenesulfonimide (NFSI) in the presence of abase like bis(trimethylsilyl) amide provides fluoro lactone (1b). Theα-chloro substituent is then conveniently introduced by reaction withN-chlorosuccinimide in the presence of a base like lithiumbis(trimethylsilyl) amide or similar. Subsequent reduction of the ketofunction using any suitable reducing agent such as DIBAL or the like,followed by conversion of the afforded hydroxy group to a leaving group,for instance a derivative of sulfonic acid, a halide or a phosphateester provides the glycosyl donor (1e). A derivative of sulfonic acidsuch as a methylsulfone, is typically prepared by treatment with mesylchloride or equivalent in the presence of a base such as Et₃N; theglycosyl bromide is typically prepared via the anomeric acetate byacetylation of the hydroxy group using acetic anhydride or similarfollowed by treatment with hydrogen bromide in acetic acid. Thenucleoside (1f) is then formed by condensation with the desired base ora protected derivative thereof using standard conditions well known inthe field of nucleoside chemistry such as in the presence ofhexamethyldisilazane (HDMS) and a Lewis acid such as TMS triflate, ortin tetrachloride or similar. In the case the glycosyl bromide is usedas glycosyl donor, a promotor for the glycosylation reaction liketintetrachloride or a silver salt such as silver triflate or similar issuitably used. Removal of the hydroxy protecting groups and, if present,protecting groups on the base, using the appropriate methods accordingto the groups by standard methods well known in the art, then providesthe nucleoside (1g). If desired, the afforded nucleoside (1g) can thenbe transformed into a 5′-mono, di- or tri-phosphate, a 5′-thio-mono-,thio-di- or thio-triphosphate, or to a prodrug using any of the methodsdescribed herein below or according to literature procedures.

Compounds of the invention carrying a cyclic phosphate ester prodrugmoiety linking the 3′-position and 5′-positions together, i.e. R¹ and R²together with the oxygen atoms to which they are attached form a cyclicphosphate ester, can be prepared for example according to the methodsdescribed in WO2010/075554. A route to such compounds wherein R³ isOR^(3′) and R^(3′) is H, C₁-C₆alkyl, C₃-C₇cycloalkyl,C₃-C₇cycloalkylC₁-C₃alkyl or benzyl, and a phosphorus(III)-reagent isused for the introduction of the phosphorus moiety, is depicted inScheme 2A.

Reaction of the diol (2a), prepared as described above with aphosphorus(III)-reagent, such asalkyl-N,N,N′,N′-tetraisopropylphosphoramidate, carrying the desiredgroup R^(3′) in the presence of an activator such as tetrazole ordicyanoimidazole or the like, provides the cyclic phosphite ester (2b).Subsequent oxidation of the phosphite ester to the phosphate ester (2c)is then carried out using any convenient oxidation method known in theart, e.g. oxidation using a peroxide reagent such as m-chloroperbenzoicacid, tert-butylhydroperoxide, hydrogen peroxide or the like.Alternatively, TEMPO-oxidation or an iodine-THF-pyridine-water basedoxidation, or any other suitable oxidation method may be used.

Similarly, the corresponding cyclic thiophosphate prodrug, i.e. U is Sin compounds of the invention carrying 3′,5′-cyclic prodrug moiety (2d),will be obtained by sulfurization of the phosphite derivative (2b).suitable sulfurization agents include, but are not limited to, elementalsulfur, Lawesson's reagent, cyclooctasulfur,bis(triethoxysilyl)propyl-tetrasulfide (TEST).

The cyclic phosphate ester (2c), may alternatively be prepared directlyin one step by reaction of the diol with a P(V)-reagent, such as alkylphosphorodichloridate, thus avoiding the separate oxidation step.

Phosphorus (III) and phosphorus r reagents to be used in the formationof the cyclic phosphite and phosphate esters respectively can beprepared as described in WO2010/075554. In short, reaction ofcommercially available chloro-N,N,N′,N′-tetraisopropylphosphoramiditewith the desired alcohol, R^(3′)—OH in the presence of a tertiary aminesuch as Et₃N provides the phosphorus (III) reagent, whereas reaction ofphosphoryl trichloride (POCl₃) with the desired alcohol R^(3′)—OH in thepresence of Et₃N or similar, provides the phosphorus (V) reagent.

Cyclic phosphate ester prodrugs of the invention wherein U is O, R³ isNHC(R¹⁵)(R^(15′))C(═O)R¹⁶, may be prepared as depicted in Scheme 2B.

Formation of the cyclic phosphate ester (2Ab) is effected for instanceby reaction of the of the diol (2a) with a phosphorylating agentcarrying the desired amino acid ester and two leaving groups (2Aa), forinstance two p-nitrophenol groups, in the presence of a base such as DBUor equivalent using a solvent such as MeCN or the like.

In a similar manner, the corresponding cyclic thiophosphate prodrug,i.e. U is S in compounds of the invention carrying a 3′,5′-cyclicprodrug moiety, will be obtained by using the corresponding thiophosphoramidate as phosphorylating agent.

For the preparation of compounds of the invention wherein R² is H and R¹is a phosphoramidate, i.e. a prodrug moiety of formula (iv), advantagecan be taken of the higher reactivity of the primary 5′-hydroxy groupcompared to the secondary 3′-hydroxy group, and the phosphoramidate canbe introduced directly on the 3′,5-diol without need of any specialprotecting group strategy. This method is illustrated in Scheme 3.

Condensation of nucleoside derivative (3a), prepared as described above,with a desired phosphoramidochloridate in an inert solvent such as anether, e.g. diethyl ether or THF, or a halogenated hydrocarbon, e.g.dichloromethane, in the presence of a base such as a N-methylimidazole(NMI) or the like, provides the phosphoramidate derivative (3b).

Similarly, compounds of the invention wherein R² is H and R¹ is athiophosphoramidate, i.e. a prodrug moiety of formula (iv) wherein U isS, are obtained by reacting the sugar (3a) with athiophosphoramidochloridate.

The phosphoramidochloridate used in the above scheme can be prepared ina two-step reaction starting from phosphorus oxychloride (POCl₃). Scheme4 illustrates the preparation of phosphoramidochloridates useful for thepreparation of compounds of formula I wherein R¹ is a group of formula(iv) wherein U is O and R²⁴ is H, and to phosphoramidochloridates usefulfor the preparation of compounds of formula I wherein R¹ is a group offormula (iv-c) wherein U is O, and R²⁴ and R^(15′) together with theatoms to which they are attached form a pyrrolidine ring.

Condensation of POCl₃ with a desired alcohol R¹⁴OH in an inert solventlike Et₂O provides alkoxy or aryloxy phosphorodichloridate (4a).Subsequent reaction with an amino acid derivative (4b) or (4b′) providesthe chlorophosphoramidate (4c) or (4c′) respectively. If desired, theobtained chlorophosphoramidates (4c) and (4c′) may be converted to thecorresponding phosphorylating agent having an activated phenol asleaving group, for instance pentaflurorophenol or p-NO₂-phenol asgenerally illustrated by FIGS. 4d and 4e respectively. This conversionis conveniently performed by reaction of the chloro derivative (4c) or(4c′) with the desired activated phenol in the presence of a base liketriethylamine or similar.

Thiophosphoramidochloridates i.e. phosphorylating reagents useful forthe preparation of compounds of formula (I) wherein R¹ is a group offormula (iv) and U is S, may be prepared using a similar strategy asgenerally outlined above, as illustrated in Scheme 5.

Reaction of thiophosphoryl chloride with a desired alcohol R¹⁴OH in thepresence of a base such as Et₃N or the like, provides alkoxy or aryloxythiophosphorodichloridate (5a). Subsequent reaction with an amino acidderivative (4b) or (4b′) provides the thiophosphoramidochloridates (5b)or (5b′) respectively.

A route to a phosphorylating agent useful for the preparation ofcompounds of formula (I) wherein R¹ is the group (v) and U is O isdepicted in Scheme 6.

Reaction of a phosphorylating agent like 4-nitrophenyl dichlorophosphatephosphoryl trichloride or similar with a suitable amine in the presenceof Et₃N or the like in a solvent like DCM, provides the desiredchlorophosphorodiamidate.

Compounds of formula (I) wherein R¹ is a prodrug moiety of group (i),R¹² and R¹³ are both R²¹(═O)S—(C₁-C₆alkylene)- and U is O, can beprepared according to literature procedures. For example, the methoddescribed in Bioorg. & Med. Chem. Let., Vol. 3, No 12, 1993, p.2521-2526, as generally illustrated in Scheme 7A.

Conversion of the 5′-hydroxy compound (7a) to the correspondinghydrogenphosphonate (7b) effected by treatment with phosphonic acid inpyridine in the presence of an activator such as pivaloyl chloride,followed by reaction with S-(2-hydroxyalkyl)alkanethioate and pivaloylchloride in pyridine and subsequent oxidation using for instanceconditions like iodine in pyridine/water provides the phosphotriester.Removal finally of protecting groups using standard methods, providesthe nucleotide prodrug (7c).

Alternatively, nucleotide prodrug (7c) may be prepared byphosphorylation of the nucleoside (7a) with a phosphorylating agentalready carrying the appropriate substituents. This method is describedin WO2013/096679 and illustrated in Scheme 7B.

Reaction of nucleoside (7a) with the phosphorylating agent, in thepresence of 5-ethylthiotetrazole (ETT), followed by oxidation using forinstance mCPBA, provides the desired prodrug (7c). The phosphorylatingagent is suitably prepared according to literature procedures asgenerally sketched out in Scheme 8.

Reaction of the desired acylchloride R²¹C(═O)Cl with mercaptoalcanol ofthe desired configuration in the presence of a tertiary amine such astriethylamine or equivalent, followed by treatment of the afforded acylthioalkanol derivative (8a) with1,1-dichloro-N,N-diisopropylphosphinamine provides the phosphorylatingagent (8b).

Compounds of formula I, wherein R¹ is a prodrug moiety of group (i) andR¹² and R¹³ are of the formula R²¹C(═O)O—C₁-C₆alkylene- orR²¹OC(═O)O—C₁-C₆alkylene- can be prepared according to the methodsdescribed in e.g. WO2013/096679 and references cited therein. The methodis briefly illustrated in Scheme 9A.

Coupling of the optionally protected nucleoside 9a with a suitablebisphosphate 9b or 9b′, preferably in the form of the ammonium salt suchas the triethylammonium salt or the like, in the presence of DIEA or thelike, using suitable coupling conditions like BOP-Cl and3-nitro-1,2,4-triazole in a solvent like THF, provides the prodrugs 9cand 9c′ respectively.

In an alternative approach to compounds of formula I wherein R¹ is aprodrug moiety of group (i) and R¹² and R¹³ are of the formulaR²¹C(═O)O—C₁-C₆alkylene- or R²¹OC(═O)O—C₁-C₆alkylene-, the nucleoside 9ais reacted with phosphorus oxychloride in a first step and subsequentlyfurther reacted with the desired with an already substitutedphosphorylating agent, as illustrated in Scheme 9B

The phosphates 9c and 9c′ are obtained by reaction of nucleoside 9a withphosphorus oxychloride in using a solvent such as triethyl phosphate,followed by reaction at elevated temperature with the desiredchloroalkyl carbonate (9b″) or ester (9b′″) in the presence of DIEA.

Compounds of formula wherein R¹ is a prodrug moiety of group (i) whereinU is O, R¹² is H and R¹³ is of the formula R²¹—O—C₁-C₆alkylene- and R²¹is C₁-C₂₄alkyl can be prepared in line with methods described in e.g. J.Med. Chem., 2006, 49, 6, p. 2010-2013 and WO2009/085267 and referencescited therein. A general method is illustrated in Scheme 10A.

Formation of the phosphorylating agent (10b) performed by reaction ofthe appropriate alkoxyalkohol (10a) with phosphorus chloride in thepresent of triethylamine using for instance diethyl ether or the like assolvent, followed by phosphotylation of the optionally protectednucleoside and finally deprotection, provides the protide (10c).

In an alternative approach to compounds of formula I wherein R¹ is aprodrug moiety of group (i) wherein U is O, R¹² is H and R¹³ is of theformula R²¹—O—C₁-C₆alkylene- and R²¹ is C₁-C₂₄alkyl, aphosphorus(III)-reagent may be used as phosphorylating agent asillustrated in Scheme 10B.

The phosphorus(III) reagent is prepared by reaction of the alkoxyalkohol(10a) with the phosphineamine (10d) in the presence of a tertiary aminesuch as DIEA or similar. Subsequent phosphorylation of the nucleosidewith the afforded phosphoramidite derivative (10e) followed by oxidationusing for instance a peroxide, such as tert-butoxy peroxide or the like,provides the nucleotide (10f). Hydrolysis of the cyanoethyl moiety andremoval of protecting groups if present, provides the desired nucleotide(10c).

Compounds of formula I, wherein R¹ is a prodrug moiety of group (vi) andR¹³ is R²¹C(═O)O—CH₂— or R²¹OC(═O)O—CH₂— can be prepared according tothe methods described in e.g. WO2013/039920 and references citedtherein. The method is briefly illustrated in Scheme 11A.

The phosphoramidates 11c an 11c′ are obtained by reaction of nucleoside11a with phosphorus oxychloride in triethyl phosphate, followed byreaction with the desired amine NHR¹⁷R^(17′) in the presence of DIEA andfinally reaction under elevated temperature with the chloroalkylcarbonate (11b) or ester (11b′) in the presence of DIEA.

Compounds of formula I, wherein R¹ is a prodrug moiety of group (vi) andR¹³ is R²¹C(═O)S—CH₂CH₂— can be prepared according to the methoddescribed in WO2008/082601 and references cited therein. The method isbriefly illustrated in Scheme 12A.

Phosphorylation of 5′-hydroxy compound (12a) with a suitable tetraalkylammonium salt, e.g. the tetraethylammonium salt, of the desired hydrogenphosphonate, effected by activation with pivaloyl chloride in pyridine,provides the hydrogen phosphonate (12b). The amino group NR¹⁷R^(17′) isthen introduced by reaction with the desired amine incarbontetrachloride under anhydrous conditions, followed by removal ofthe protecting groups, thus yielding the phosphoramidate (12c).

As an alternative, phosphoramidate (12c) can be achieved from theH-phosphonate (7b) of Scheme 7A by reaction with a desiredS-(2-hydroxyethyl) alkanethioate R²¹C(C═O)SCH₂CH₂OH, in the presence ofa coupling agent such as PyBOP or the like, followed by amination anddeprotection as described above. This route is illustrated in Scheme12B.

As the skilled person will realise, the procedures illustrated inSchemes 12A and 12B will be applicable not only for the preparation ofS-acylthioethanol derivatives, but also of derivatives having otheralkylene configurations between the sulfur and oxygen atoms.

Compounds of the invention having an acyl prodrug moiety in the5′-position and optionally also in the 3′-position, i.e. R¹ andoptionally also R² are C(O═)R³⁰ or C(═O)R³¹NH₂ can be obtained bysubjection of a suitably 3′-protected compound to suitable acylatingconditions, as illustrated in Scheme 13.

Nucleoside (13b) wherein the prodrug group in the 5′-position is anester i.e. a group of the formula OC(═O)R¹⁰, is obtained by reaction of5′-hydroxy compound (9a) with the appropriate acylating agent usingstandard methods, such as using an alkyl acid anhydride,R³⁰C(═O)OC(═O)R³⁰, in the presence of pyridine, or an alkyl acidchloride, R³⁰C(═O)Cl, or the like, whereas nucleosides (13d) carrying anamino acid ester in the 5′-position will be obtained by reaction of the5′-hydroxy compound (13a) with an N-protected aliphatic amino acid inthe presence of a suitable peptide coupling reagent such as EDAC or thelike. Removal of the 3′-hydroxy protecting group then yields compoundsof the invention wherein R² is H. On the other hand, subjection of the3′-hydroxy compounds (13b) and (13c) to the acylation conditionsdescribed immediately above, yields the diacyl derivatives (13d) and(13e) respectively.

Compounds of the invention carrying an ester or amino acid ester prodrugmoiety in the and/or 3′-position may be prepared as illustrated inScheme 14.

Due to the higher reactivity of the primary 5′-position of the diol(14a), this position can be selectively reacted with a suitableacylating agent to obtain 5′-acyl derivatives (14b) and (14c), or it canbe protected with a suitable protecting group to allow for subsequentacylation of the 3′-position. Nucleosides (14b) wherein the prodruggroup in the 5′-position is an ester i.e. a group of the formulaOC(═O)R³⁰, are conveniently obtained by reaction with acylating agentsuch as an alkyl anhydride in the presence of pyridine, or an acidchloride or the like, whereas nucleosides (14c) carrying an amino acidester in the 5′-position will be obtained by reaction of the diol (14a)with an N-protected aliphatic amino acid in the presence of a suitablepeptide coupling reagent such as EDAC or the like. If an acyl prodruggroup is desired in the 3′-position, a protection-acylation-deprotectionsequence will be appropriate in order to get clean reactions withdescent yields. Typically, a protecting group like a silyl, trityl or amonomethoxy trityl (MMT) group will be suitable to protect the5′-hydroxy group. The use of these groups are extensively described inthe literature, typically, conditions like reaction with thecorresponding halide, such as the chloride in a solvent like pyridine isused for their introduction. Subsequent acylation performed as describedabove, followed by removal of the 5′-O-protecting group, and in case ofthe amino acid ester being introduced as an N-protected amino acid, theN-protecting group, using the appropriate conditions according to theprotecting group used, such as acidic treatment in the case of a tritylor methoxy trityl protecting group, then provides the 3′-acylatedderivatives (14d) and (14e). If desired, a phosphoramidate can beintroduced in the 5′-position of the afforded 5′-hydroxy derivatives(14d) and (14e), for example using the procedure described herein above,or a mono-, di- or tri-phosphate may be introduced using standardliterature phosphorylation procedures, or the 5′-position may beacylated using the method described above for acylation of the3′-position.

Compounds of the invention having an acetal prodrug moiety in the5′-position or in both the 5′- and 3′-positions, i.e. compounds offormula I wherein R¹ or both R¹ and R² is CR³²R^(32′)OC(═O)CHR³³NH₂ canbe prepared from the 5′-hydroxy compound using for example the methoddescribed in Bioorg. Med. Chem. 11(2003) 2453-2461.

Compounds of the invention carrying a “HepDirect” prodrug moiety in the5′-position, i.e. a compound of formula I wherein R¹ is the group (i),and R¹² and R¹³ join to form a propylene group between the oxygen atomsto which they are attached, can be prepared according to the methoddescribed in J. Am. Chem. Soc., Vol. 126, No. 16, 2004, p. 5154-5163.

A route to compounds of formula I wherein B is the group (a) or (b), R²is H and R¹ is a triphosphate, i.e. a group of formula (iii), wherein Uis O, is illustrated in Scheme 15.

A suitable phosphorylating agent for the preparation of the triphosphateof the compounds of formula (I) wherein B is the group (a) or (b) is5-nitrocyclosalgenylchlorophosphite (I-6), which is prepared by reactionof phosphorous trichloride and 2-hydroxy-5-nitrobenzyl alcohol asdetailed in the experimental part herein below.

Reaction of a suitably 3′-O-protected derivative of the nucleoside ofthe invention (15a) with nitrocyclosalgenylchlorophosphite (I-1) in thepresence of Et₃N in an inert solvent like DCM or MeCN, followed byoxidation using for instance Oxone®, provides the cyclic phosphatetri-ester (15b). The triphosphate (15c) is then achieved by reactionwith a pyrophosphate for instance tributylamine pyrophosphate followedby treatment with ammonia. In order to get the desired salt form, thetriphosphate is subjected to the appropriate ion exchange procedure, forinstance, if the potassium salt form is desired, the residue is passedthrough a column Dowex®-K⁺.

A route to compounds of formula I wherein B is uracil, R² is H and R¹ isa thio-triphosphate, i.e. a group of formula (iii), wherein U is S, isillustrated in Scheme 16.

A suitable agent for introduction of the first phosphate group in thepreparation of a thio-triphosphate of the U-nucleoside of a compound offormula (I) is 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, which isprepared according to literature procedures. A suitably 3′-O-protectednucleoside is thus reacted with2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one in a solvent likepyridine/THF or equivalent followed by treatment with tributylammoniumpyrophosphate in the presence of tributylamine in a solvent like DMF.The afforded intermediate is then transformed to the thiotriphosphate bytreatment with a solution of sulfur in DMF. In order to get the desiredsalt form, the triphosphate is subjected to the appropriate ion exchangeprocedure, for instance, if the lithium salt form is desired, theresidue is passed through a column Dowex®-Li⁺.

An alternative route to the thio-triphosphate is illustrated in Scheme17.

In this method, a thiophosphate reagent is used in the phosphorylationstep. The reagent is prepared by reaction of PSCl₃ and triazole in asolvent like MeCN or similar. The thus formed reagent is then coupled tothe 3′-O-protected nucleoside 13a, whereafter a reaction with apyrophosphate such as tris(tetrabutylammonium) hydrogen pyrophosphate isperformed, thus providing the thio-triphosphate (17b).

The use of various protecting groups (PG) used in schemes above areknown to the skilled person, and their utility and further alternativesare extensively described in the literature, see for instance Greene T.W., Wuts P. G. M. Protective groups in organic synthesis, 2nd ed. NewYork: Wiley; 1995.

The term “N-protecting group” or “N-protected” as used herein refers tothose groups intended to protect the N-terminus of an amino acid orpeptide or to protect an amino group against undesirable reactionsduring synthetic procedures. Commonly used N-protecting groups aredisclosed in Greene. N-protecting groups include acyl groups such asformyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl,2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl,o-nitrophenoxyacetyl; α-chlorobutyryl, benzoyl, 4-chlorobenzoyl,4-bromobenzoyl; 4-nitrobenzoyl, and the like; sulfonyl groups such asbenzenesulfonyl, p-toluenesulfonyl, and the like; carbamate forminggroups such as benzyloxycarbonyl, p-chlorobenzyloxy-carbonyl,p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,3,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl;ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike; alkyl groups such as benzyl, triphenyl methyl, benzyloxymethyl andthe like; and silyl groups such as trimethylsilyl and the like. FavouredN-protecting groups include formyl, acetyl, benzoyl, t-butylacetyl,phenylsulfonyl, benzyl (Bz), t-butoxycarbonyl (BOC) andbenzyloxycarbonyl (Cbz).

Hydroxy and/or carboxy protecting groups are also extensively reviewedin Greene ibid and include ethers such as methyl, substituted methylethers such as methoxymethyl, methylthiomethyl, benzyloxymethyl,t-butoxymethyl, 2-methoxyethoxymethyl and the like, silyl ethers such astrimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS) tribenzylsilyl,triphenylsilyl, t-butyldiphenylsilyl, triisopropyl silyl and the like,substituted ethyl ethers such as 1-ethoxymethyl,1-methyl-1-methoxyethyl, t-butyl, benzyl, p-methoxybenzyl,diphenylmethyl, triphenylmethyl and the like, aralkyl groups such astrityl, and pixyl (9-hydroxy-9-phenylxanthene derivatives, especiallythe chloride). Ester hydroxy protecting groups include esters such asformate, benzylformate, chloroacetate, methoxyacetate, phenoxyacetate,pivaloate, adamantoate, mesitoate, benzoate and the like. Carbonatehydroxy protecting groups include methyl vinyl, allyl, cinnamyl, benzyland the like.

In one aspect, the present invention concerns a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof formula I, and a pharmaceutically acceptable carrier. Atherapeutically effective amount in this context is an amount sufficientto stabilize or to reduce viral infection, and in particular HCVinfection, in infected subjects (e.g. humans). The “therapeuticallyeffective amount” will vary depending on individual requirements in eachparticular case. Features that influence the dose are e.g. the severityof the disease to be treated, age, weight, general health condition etc.of the subject to be treated, route and form of administration.

In one aspect, the invention relates to the use of a compound of formulaI, for the treatment of “treatment naive” patients, i.e. patientsinfected with HCV that are not previously treated against the infection.

In another aspect the invention relates to the use of a compound offormula I, the treatment of “treatment experienced” patients, i.e.patients infected with HCV that are previously treated against theinfection and have subsequently relapsed.

In another aspect the invention relates to the use of a compound offormula I, the treatment of “non-responders”, i.e. patients infectedwith HCV that are previously treated but have failed to respond to thetreatment.

In a further aspect, the present invention concerns a pharmaceuticalcomposition comprising a prophylactically effective amount of a compoundof formula as specified herein, and a pharmaceutically acceptablecarrier. A prophylactically effective amount in this context is anamount sufficient to act in a prophylactic way against HCV infection, insubjects being at risk of being infected.

In still a further aspect, this invention relates to a process ofpreparing a pharmaceutical composition as specified herein, whichcomprises intimately mixing a pharmaceutically acceptable carrier with atherapeutically or prophylactically effective amount of a compound offormula I, as specified herein.

Therefore, the compounds of the present invention may be formulated intovarious pharmaceutical forms for administration purposes. As appropriatecompositions there may be cited all compositions usually employed forsystemically administering drugs. To prepare the pharmaceuticalcompositions of this invention, an effective amount of the particularcompound, optionally in addition salt form or solvate, as the activeingredient is combined in intimate admixture with a pharmaceuticallyacceptable carrier, which carrier may take a wide variety of formsdepending on the form of preparation desired for administration. Thesepharmaceutical compositions are desirable in unitary dosage formsuitable, particularly, for administration orally, rectally,percutaneously, or by parenteral injection. For example, in preparingthe compositions in oral dosage form, any of the usual pharmaceuticalmedia may be employed such as, for example, water, glycols, oils,alcohols and the like in the case of oral liquid preparations such assuspensions, syrups, elixirs, emulsions and solutions; or solid carrierssuch as starches, sugars, kaolin, lubricants, binders, disintegratingagents and the like in the case of powders, pills, capsules, andtablets. Because of their ease in administration, tablets and capsulesrepresent the most advantageous oral dosage unit forms, in which casesolid pharmaceutical carriers are obviously employed. For parenteralcompositions, the carrier will usually comprise sterile water, at leastin large part, though other ingredients, for example, to aid solubility,may be included, injectable solutions, for example, may be prepared inwhich the carrier comprises saline solution, glucose solution or amixture of saline and glucose solution. Injectable suspensions may alsobe prepared in which case appropriate liquid carriers, suspending agentsand the like may be employed. Also included are solid form preparationsintended to be converted, shortly before use, to liquid formpreparations. In the compositions suitable for percutaneousadministration, the carrier optionally comprises a penetration enhancingagent and/or a suitable wetting agent, optionally combined with suitableadditives of any nature in minor proportions, which additives do notintroduce a significant deleterious effect on the skin. The compounds ofthe present invention may also be administered via oral inhalation orinsufflation in the form of a solution, a suspension or a dry powderusing any art-known delivery system.

It is especially advantageous to formulate the aforementionedpharmaceutical compositions in unit dosage form for ease ofadministration and uniformity of dosage. Unit dosage form as used hereinrefers to physically discrete units suitable as unitary dosages, eachunit containing a predetermined quantity of active ingredient calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. Examples of such unit dosage forms aretablets (including scored or coated tablets), capsules, pills,suppositories, powder packets, wafers, injectable solutions orsuspensions and the like, and segregated multiples thereof.

The compounds of formula I show activity against HCV and can be used inthe treatment and/or prophylaxis of HCV infection or diseases associatedwith HCV. Typically the compounds of formula I can be used in thetreatment of HCV infection or diseases associated with HCV. Diseasesassociated with HCV include progressive liver fibrosis, inflammation andnecrosis leading to cirrhosis, end-stage liver disease, and HCC. Anumber of the compounds of this invention may be active against mutatedstrains of HCV. Additionally, many of the compounds of this inventionmay show a favourable pharmacokinetic profile and have attractiveproperties in terms of bioavailability, including an acceptablehalf-life, AUC (area under the curve) and peak values and lackingunfavourable phenomena such as insufficient quick onset and tissueretention.

The in vitro antiviral activity against HCV of the compounds of formulaI can be tested in a cellular HCV replicon system based on Lohmann etal. (1999) Science 285:110-113, with the further modifications describedby Krieger et al, (2001) Journal of Virology 75: 4614-4624 (incorporatedherein by reference), which is further exemplified in the examplessection. This model. While not a complete infection model for HCV, iswidely accepted as the most robust and efficient model of autonomous HCVRNA replication currently available. It will be appreciated that it isimportant to distinguish between compounds that specifically interferewith HCV functions from those that exert cytotoxic or cytostatic effectsin the HCV replicon model, and as a consequence cause a decrease in HCVRNA or linked reporter enzyme concentration. Assays are known in thefield for the evaluation of cellular cytotoxicity based for example onthe activity of mitochondrial enzymes using fluorogenic redox dyes suchas resazurin. Furthermore, cellular counter screens exist for theevaluation of non-selective inhibition of linked reporter gene activity,such as firefly luciferase. Appropriate cell types can be equipped bystable transfection with a luciferase reporter gene whose expression isdependent on a constitutively active gene promoter, and such cells canbe used as a counter-screen to eliminate non-selective inhibitors.

Due to their antiviral properties, particularly their anti-HCVproperties, the compounds of formula I, including any possiblestereoisomers, the pharmaceutically acceptable addition salts orsolvates thereof, are useful in the treatment of warm-blooded animals,in particular humans, infected with HCV. The compounds of formula I arefurther useful for the prophylaxis of HCV infections. The presentinvention furthermore relates to a method of treating a warm-bloodedanimal, in particular human, infected by HCV, or being at risk ofinfection by HCV, said method comprising the administration of ananti-HCV effective amount of a compound of formula I.

The compounds of the present invention may therefore be used as amedicine, in particular as an anti HCV medicine. Said use as a medicineor method of treatment comprises the systemic administration to HCVinfected subjects or to subjects susceptible to HCV infection of anamount effective to combat the conditions associated with HCV infection.

The present invention also relates to the use of the present compoundsin the manufacture of a medicament for the treatment or the preventionof HCV infection.

In a preferred embodiment, the present invention relates to the use ofthe compounds of formula I in the manufacture of a medicament for thetreatment of HCV infection.

In general it is contemplated that an antiviral effective daily amountwould be from about 0.01 to about 700 mg/kg, or about 0.5 to about 400mg/kg, or about 1 to about 250 mg/kg, or about 2 to about 200 mg/kg, orabout 10 to about 150 mg/kg body weight. It may be appropriate toadminister the required dose as two, three, four or more sub-doses atappropriate intervals throughout the day. Said sub-doses may beformulated as unit dosage forms, for example, containing about 1 toabout 5000 mg, or about 50 to about 3000 mg, or about 100 to about 1000mg, or about 200 to about 600 mg, or about 100 to about 400 mg of activeingredient per unit dosage form.

The invention also relates to a combination of a compound of formula I,a pharmaceutically acceptable salt or solvate thereof, and anotherantiviral compound, in particular another anti-HCV compound. The term“combination” may relate to a product containing (a) a compound offormula I and (b) optionally another anti-HCV compound, as a combinedpreparation for simultaneous, separate or sequential use in treatment ofHCV infections.

Anti-HCV compounds that can be used in such combinations include HCVpolymerase inhibitors, HCV protease inhibitors, inhibitors of othertargets in the HCV life cycle, and an immunomodulatory agents, andcombinations thereof, HCV polymerase inhibitors include, NM283(valopicitabine), R803, JTK-109, JTK-003, HCV-371, HCV-086, HCV-796 andR-1479, R-7128, MK-0608, VCH-759, PF-868554, GS9190, XTL-2125, NM-107,GSK625433, R-1626, BILB-1941, ANA-598, IDX-184, IDX-375, INX-189,MK-3281, MK-1220, ABT-333, PSI-7851, PSI-6130, GS-7977 (sofosbuvir),VCH-916. Inhibitors of HCV proteases (NS2-NS3 inhibitors and NS3-NS4Ainhibitors) include BILN-2061, VX-950 (telaprevir), GS-9132 (ACH-806),SCH-503034 (boceprevir), TMC435350 (simeprevir), TMC493706, ITMN-191,MK-7009, BI-12202, BILN-2065, BI-201335, BMS-605339, R-7227, VX-500,BMS650032, VBY-376, VX-813, SCH-6, PHX-1766, ACH-1625, IDX-136, IDX-316.An example of an HCV NS5A inhibitor is BMS790052, A-831, A-689, NIM-811and DEBIO-025 are examples of NS5B cyclophilin inhibitors.

Inhibitors of other targets in the HCV life cycle, including NS3helicase; metalloprotease inhibitors; antisense oligonucleotideinhibitors, such as ISIS-14803 and AVI-4065; siRNA's such asSIRPLEX-140-N; vector-encoded short hairpin RNA (shRNA); DNAzymes; HCVspecific ribozymes such as heptazyme, RPI. 13919; entry inhibitors suchas HepeX-C, HuMax-HepC; alpha glucosidase inhibitors such as celgosivir,UT-231B and the like; KPE-02003002; and BIVN 401.

Immunomodulatory agents include, natural and recombinant interferonisoform compounds, including α-interferon, β-interferon, γ-interferon,and ω-interferon, such as Intron A®, Roferon-A®, Canferon-A300®,Advaferon®, Infergen®, Humoferon®, Sumiferon MP®, Alfaferone®,IFN-beta®, and Feron®; polyethylene glycol derivatized (pegylated)interferon compounds, such as PEG interferon-α-2a (Pegasys®), PEGinterferon-α-2b (PEG-Intron®), and pegylated IFN-α-con1; long actingformulations and derivatizations of interferon compounds such as thealbumin-fused interferon albuferon α; compounds that stimulate thesynthesis of interferon in cells, such as resiquimod; interleukins;compounds that enhance the development of type 1 helper T cell response,such as SCV-07; TOLL-like receptor agonists such as CpG-10101 (actilon),and isatoribine; thymosin α-1; ANA-245; ANA-246; histaminedihydrochloride; propagermanium; tetrachlorodecaoxide; ampligen;IMP-321; KRN-7000; antibodies, such as civacir and XTL-6865; andprophylactic and therapeutic vaccines such as InnoVac C and HCVE1E2/MF59.

Other antiviral agents include, ribavirin, amantadine, viramidine,nitazoxanide; telbivudine; NOV-205; taribavirin; inhibitors of internalribosome entry; broad-spectrum viral inhibitors, such as IMPDHinhibitors, and mycophenolic acid and derivatives thereof, andincluding, but not limited to, VX-497 (merimepodib), VX-148, and/orVX-944); or combinations of any of the above.

Particular agents for use in said combinations include interferon-α(IFN-α), pegylated interferon-α or ribavirin, as well as therapeuticsbased on antibodies targeted against HCV epitopes, small interfering RNA(Si RNA), ribozymes, DNAzymes, antisense RNA, small molecule antagonistsof for instance NS3 protease, NS3 helicase and NS5B polymerase.

In another aspect there are provided combinations of a compound offormula I as specified herein and an anti-HIV compound. The latterpreferably are those HIV inhibitors that have a positive effect on drugmetabolism and/or pharmacokinetics that improve bioavailability. Anexample of such an HIV inhibitor is ritonavir. As such, this inventionfurther provides a combination comprising (a) a compound of formula I ora pharmaceutically acceptable salt or solvate thereof; and (b) ritonaviror a pharmaceutically acceptable salt thereof. The compound ritonavir,its pharmaceutically acceptable salts, and methods for its preparationare described in WO 94/14436. U.S. Pat. No. 6,037,157, and referencescited therein: U.S. Pat. No. 5,484,801, U.S. Ser. No. 08/402,690, WO95/07696, and WO 95/09614, disclose preferred dosage forms of ritonavir.

The invention also concerns a process for preparing a combination asdescribed herein, comprising the step of combining a compound of formulaI and another agent, such as an antiviral, including an anti-HCV oranti-HIV agent, in particular those mentioned above.

The said combinations may find use in the manufacture of a medicamentfor treating HCV infection in a mammal infected therewith, saidcombination in particular comprising a compound of formula as specifiedabove and interferon-α (IFN-α), pegylated interferon-α, or ribavirin. Orthe invention provides a method of treating a mammal, in particular ahuman, infected with HCV comprising the administration to said mammal ofan effective amount of a combination as specified herein. In particular,said treating comprises the systemic administration of the saidcombination, and an effective amount is such amount that is effective intreating the clinical conditions associated with HCV infection.

In one embodiment the above-mentioned combinations are formulated in theform of a pharmaceutical composition that includes the activeingredients described above and a carrier, as described above. Each ofthe active ingredients may be formulated separately and the formulationsmay be co-administered, or one formulation containing both and ifdesired further active ingredients may be provided. In the formerinstance, the combinations may also be formulated as a combinedpreparation for simultaneous, separate or sequential use in HCV therapy.The said composition may take any of the forms described above. In oneembodiment, both ingredients are formulated in one dosage form such as afixed dosage combination. In a particular embodiment, the presentinvention provides a pharmaceutical composition comprising (a) atherapeutically effective amount of a compound of formula I, including apossible stereoisomeric form thereof, or a pharmaceutically acceptablesalt thereof, or a pharmaceutically acceptable solvate thereof, and (b)a therapeutically effective amount of ritonavir or a pharmaceuticallyacceptable salt thereof, and (c) a carrier.

The individual components of the combinations of the present inventioncan be administered separately at different times during the course oftherapy or concurrently in divided or single combination forms. Thepresent invention is meant to embrace all such regimes of simultaneousor alternating treatment and the tem “administering” is to beinterpreted accordingly. In a preferred embodiment, the separate dosageforms are administered simultaneously.

In one embodiment, the combinations of the present invention contain anamount of ritonavir, or a pharmaceutically acceptable salt thereof, thatis sufficient to clinically improve the bioavailability of the compoundof formula I relative to the bioavailability when said compound offormula I is administered alone. Or, the combinations of the presentinvention contains an amount of ritonavir, or a pharmaceuticallyacceptable salt thereof, which is sufficient to increase at least one ofthe pharmacokinetic variables of the compound of formula I selected fromt_(1/2), C_(min), C_(max), C_(ss), AUC at 12 hours, or AUC at 24 hours,relative to said at least one pharmacokinetic variable when the compoundof formula I is administered alone.

The combinations of this invention can be administered to humans indosage ranges specific for each component comprised in saidcombinations, e.g. the compound of formula I as specified above, andritonavir or a pharmaceutically acceptable salt, may have dosage levelsin the range of 0.02 to 5.0 g/day.

The weight ratio of the compound of formula I to ritonavir may be in therange of from about 30:1 to about 1:15, or about 15:1 to about 1:10, orabout 15:1 to about 1:1, or about 10:1 to about 1:1, or about 8:1 toabout 1:1, or about 5:1 to about 1:1, or about 3:1 to about 1:1, orabout 2:1 to 1:1. The compound formula I and ritonavir may beco-administered once or twice a day, preferably orally, wherein theamount of the compound of formula I per dose is as described above; andthe amount: of ritonavir per dose is from 1 to about 2500 mg, or about50 to about 1500 mg, or about 100 to about 800 mg, or about 100 to about400 mg, or 40 to about 100 mg of ritonavir.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Various embodiments of the invention and intermediates therefore willnow be illustrated by the following examples. The Examples are justintended to further illustrate the invention and are by no meanslimiting the scope of the invention. The compound names were generatedby ChemDraw Ultra software, Cambridgesoft, version 12.0.2.

In addition to the definitions above, the following abbreviations areused in the examples and synthetic schemes below. If an abbreviationused herein is not defined, it has its generally accepted meaning

-   Bn Benzyl-   Bz Benzoyl-   BOP-Cl Bis(2-oxo-3-oxazolidinyl)phosphinic chloride-   Bz Benzoyl-   DCC Dicyclohexylcarbodiimide-   DCM Dichloromethane-   DIEA Diisopropylethylamine-   DMAP 4-Dimethylaminopyridine-   DMF N,N-Dimethylformamide-   DMPU 1,3-Dimethyl-3,4,5,6-tetrahydro-2-pyrimidinone-   EDC 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride-   ES Electrospray-   Et₃N Triethylamine-   EtOAc Ethyl acetate-   EtOH Ethanol-   Et₂O Diethyl ether-   LC Liquid chromatography-   HOAc Acetic acid-   HPLC High performance liquid chromatography-   MeCN Acetonitrile-   MeOH Methanol-   MS Mass spectrometry-   NT 3-nitro-1,2,4-triazole-   NTP Nucleoside triphosphate-   Pg Protecting group-   Ph Phenyl-   SEM Standard error of the mean-   TEST bis(triethoxysilyl)propyl-tetrasulfide-   THF Tetrahydrofuran-   TFA Trifluoroacetic acid-   TFAA Trifluoroacetic anhydride-   TIPS Triisopropylsilyl

The following phenols were prepared and used in the preparation ofintermediates to the compounds of the invention:

Phenol 1

Step a) 1-(3-((Tert-butyldimethylsilyl)oxy)phenyl)ethanone (Ph1-a)

Imidazole (4.46 g, 65.5 mmol) was added to a solution of3-hydroxyacetophenone (4.46 g, 32.8 mmol) in DMF (6 mL). After 5 min, asolution of TBDMS-Cl (4.69 g, 31.1 mmol) in DMF (4 mL) was added. Thereaction mixture was stirred at room temperature for 90 min, then pouredinto hexane containing 5% EtOAc (200 mL) and washed with 1M HCl (60 mL),water (60 mL), saturated sodium bicarbonate (2×60 mL), water (60 mL) andbrine (60 mL). The organic layer was dried over Na₂SO₄, filtered andconcentrated and the afforded residue was purified by flashchromatography on silica gel eluted with hexane/EtOAc, which gave thetitle compound (5.7 g, 69%).

Step b) Tert-butyldimethyl(3-(prop-1-en-2-yl)phenoxy)silane (Ph1-b)

Methyl(triphenylphosphonium)bromide (10.2 g, 28.4 mmol) was suspended indry THF (30 mL) under nitrogen and the suspension was cooled to 0° C.n-Butyllithium (17.8 mL, 28.4 mmol) was added drop-wise to the mixtureand the resulting solution was stirred at room temperature for 30 min.Ph1-a (5.7 g, 22.8 mmol) was added to the mixture and the reactionallowed to proceed at room temperature for 60 min. The reaction wasquenched with aqueous sodium bicarbonate and extracted with diethylether (50 mL). The organic layer was washed with sodium bicarbonatesolution, dried (Na₂SO₄), filtered and concentrated. The affordedresidue was purified through a plug of silica-gel using eluted withhexane, which gave the title compound (3.9 g, 69%).

Step c) Tert-butyldimethyl(3-(1-methylcyclopropyl)phenoxy)silane (Ph1-c)

Diethylzinc in hexane (439.2 mmol) was added drop-wise under nitrogenduring 10 minutes to a cooled (0° C.) solution of the olefin Ph1-b (3.9g, 15.7 mmol) in 1,2-dichloroethane (60 mL). Diiodomethane (6.32 mL,78.5 mmol) was added drop-wise and the resulting mixture was stirred at0° C. for 30 min and then allowed to attain room temperature overnight.The mixture was poured into an ice-cold solution of ammonium chlorideand extracted with diethyl ether. The organic layer was washed withsaturated sodium bicarbonate, dried (Na₂SO₄), filtered and concentrated.The crude was taken into hexane and the remaining diiodomethane wasdiscarded. The hexane layer was concentrated to a crude that was takeninto the next step without further purification.

Step d) 3-(1-Methylcyclopropyl)phenol (Phenol 1)

Ph1-c (3.45 g, 13.1 mmol) was taken into 1M solution oftetrabutylammonium fluoride in THF (20 mL, 20 mmol) and the resultingsolution was stirred at room temperature overnight. The reaction wasquenched with 1M HCl (50 mL) and extracted with ethyl acetate (100 mL).The organic layer was washed with brine (2×50 mL), dried (Na₂SO₄),filtered and concentrated. The residue was purified by flashchromatography on silica gel eluted with a mixture of 2-propanol, EtOAcand hexane, which gave the title compound (0.56 g, 29%). MS 147.1[M−H]⁻.

Phenol 2

The title compound was prepared from 4-hydroxyacetophenone (6.0 g, 44.1mmol) using the method described for the preparation of Phenol 1. Yield53%.

Phenol 3

Step a) 1-(3-(benzyloxy)phenyl)cyclopentanol (Ph3-a)

Iodine, warmed up with magnesium, was added to a suspension of magnesiumtunings (1.29 g, 52.8 mmol) in dry THF (50 mL). The mixture was refluxedand about 5% of a solution of 3-bromophenol (13.9 g, 52.8 mmol) wasadded. When the reaction had started, the solution of the bromide wasadded drop-wise and the mixture was then refluxed for one more hour. Themixture was cooled down to about 5° C. and a solution of thecyclopentanone (4.44 g, 52.8 mmol) in THF (50 mL) was added drop-wise.The mixture was stirred at rt for 72 h, then the reaction was quenchedwith cooled saturated ammonium chloride solution and extracted withdiethyl ether (×3). The organic phase was washed with brine, dried(Na₂SO₄), filtered and concentrated. The product was purified by silicagel chromatography (isohexane/EtOAc), which gave the title compound (8.5g, 54%).

Step b) 1-(benzyloxy)-3-(cyclopent-1-en-1-yl)benzene (Ph3-b)

p-Toluenesulfonic acid was added to a solution of Ph3-a (8.4 g, 28.2mmol) in benzene (100 mL). The mixture was refluxed for three hours witha DMF trap, then cooled to rt, diluted with diethyl ether and washedwith a saturated solution of sodium hydrogen carbonate and brine. Theorganic phase was dried (Na₂SO₄), filtered and concentrated. The productwas purified by silica gel chromatography (isohexane/EtOAc), which gavethe title compound (6.45 g, 91%). MS 249.4 [M−H]⁻.

Step c) 3-Cyclopentylphenol (Phenol 3)

A solution of Ph3-b (6.4 g, 26 mmol) in EtOAc (75 mL) and EtOH (75 mL)was hydrogenated at 22° C. and 40 PSI in the presence of 106 Pd oncarbon (1.5 g) in a Parr overnight. The catalyst was filtered off andwashed with EtOAc and EtOH. The solvent was evaporated under reducedpressure and the product was isolated by silica gel chromatography(isohexane/EtOAc), which gave the title compound (3.6 g, 82%). MS 161.2[M−H]⁻.

Phenol 4

Step a) Tert-butyl(3-cyclopropylphenoxy)dimethylsilane (Ph4-a)

A suspension of (3-bromophenoxy)(tert-butyl)dimethylsilane (5.46 g, 19mmol), cyclopropylboronic acid (2.12 g, 24.7 mmol), potassium phosphate,tribasic (14.1 g, 66.5 mmol), tricyclohexylphosphine (0.53 g, 1.9 mmol)and Pd(OAc)₂ (0.21 g, 0.95 mmol) in toluene (80 mL) and water (4 mL) wasstirred at 110° C. overnight. The slurry was diluted with diethyl etherand washed with water and brine. The organic phase was dried (MgSO₄),filtered and concentrated. The crude was purified by flash columnchromatography (EtOAc/hexane) which gave the title compound (1.94 g,41%).

Step b) 3-Cycloproylphenol (Phenol 4)

1M tetrabutylammonium fluoride (10.1 ml, 10.1 mmol) was added to asolution of Ph4-a (1.94 g, 7.81 mmol) in THF (25 ml). The solution wasstirred for 2 hours, then the solvent was evaporated and the residuedissolved in EtOAc and washed twice with concentrated NH₄Cl (aq) andonce with brine. The organic phase was dried (MgSO₄), filtered andconcentrated. The crude was purified by flash column chromatography(hexane/ethyl acetate 9:1 with 1% isopropanol) which gave slightlyimpure title compound (1.24 g, 119%).

Phenol 5

Step a) 2-(4-Bromophenoxy)tetrahydro-2H-pyran (Ph5-a)

4-Bromphenol (3.75 g, 21.7 mmol) was dissolved in 3,4-dihydro-2H-pyran(16 ml, 175 mmol), a catalytic amount of p-Toluenesulfonic acid (15 mg,0.09 mmol) was added and the mixture was stirred at 22° C. for 45 min.The mixture was diluted with diethyl ether and washed with 1 M NaOH(aq)×2, water, dried (Na₂SO₄) and concentrated which gave the titlecompound (5.57 g, 99%).

Step b) 2-(4-Cyclopropylphenoxy)tetrahydro-2H-pyran (Ph5-b)

A solution of 0.5 M cyclopropyl magnesium bromide in THF (6.5 ml, 3.25mmol) was added during 15 min to a solution of Ph5-a (552.5 mg, 2.15mmol), ZnBr (144 mg, 0.64 mmol), tri-tert-butylphosphinetetrafluoroborate (35.6 mg, 0.12 mmol) and Pd(OAc)₂ (29.5 mg, 0.13 mmol)in THF (4 ml). The mixture was stirred at 22° C. for 90 min then cooledon an ice bath and ice water (10 ml) was added. The mixture wasextracted with EtOAc×3 and the extracts washed with brine and then dried(Na₂SO₄), filtered and concentrated. The residue was purified by columnchromatography on silica (petroleum ether/EtOAc) which gave the titlecompound (292 mg, 62%).

Step c) 4-Cyclopropylphenol (Phenol 5)

p-Toluenesulfonic acid monohydrate (18.9 mg, 0.1 mmol) was added to asolution of Ph5-b (2.28 g, 10.45 mmol) in MeOH (15 ml). The mixture washeated at 120° C. for 5 min in a microwave reactor, then concentratedand purified by column chromatography on silica (petroleum ether/EtOAc).The afforded solids were crystallized from petroleum ether which gavethe title compound (1.08 g, 77%).

Phenol 6

Step a) 1-(3-Methoxyphenyl)cyclobutanol (Ph6-a)

A 1 M solution of 3-methoxyphenyl magnesium bromide in THF (2.11 g, 99.8mmol) was added dropwise between 0 and 10° C. to a stirred solution ofcyclobutanone (6.66 g, 95 mmol) in diethyl ether (65 mL). The mixturewas stirred for three hours at 0-10° C., then the mixture was added toan ice cooled solution of saturated NH₄Cl (300 mL) and water (300 mL).The mixture was stirred for 10 min then extracted three times withdiethyl ether. The organic phase was dried, (Na₂SO₄), filtered andconcentrate. The afforded crude product was purified by silica gelchromatography (isohexane/EtOAc), which gave the title compound (16.9 g,86%).

Step b) 1-cyclobutyl-3-methoxybenzene (Ph6-b)

10% Pd on carbon (2.5 g) was added to a solution of Ph6-a (15.4 g, 86.1mmol) in ethanol (200 mL) and the mixture was hydrogenated in a Parr at60 psi. After 18 h, additional 10% Pd on carbon (1.5 g) was added andthe mixture was hydrogenated for further 18 hours at 60 psi. Thecatalyst was filtered of and washed with EtOH and EtOAc. The solutionwas concentrated under reduced pressure and the crude product wasisolated by silica gel chromatography (isohexane/EtOAc), which gave thetitle compound (14.0 g, 77%).

Step c) 3-cyclobutylphenol (Phenol 6)

A solution of 1M boron tribromide (18.1 g, 72.2 mmol) in DCM was addeddropwise at 0° C. to a solution of Ph6-b (10.6 g, 65.6 mmol) in dry DCM(65 mL). The mixture was stirred for 2.5 hours at −5° C., then thereaction was quenched with cooled saturated solution of NH₄Cl andextracted three times with DCM. The organic phase was dried (Na₂SO₄),filtered and concentrate. The afforded crude product was purified bysilica gel chromatography (isohexane/EtOAc), which gave the titlecompound (9.73 g, 88%).

Phenol 7

Step a) 1-(4-(benzyloxy)phenyl)cyclobutanol (Ph7-a)

A solution of 1-(benzyloxy)-4-bromobenzene (2.63 g, 100 mmol) in diethylether:THF 1:1 (100 mL) was added dropwise at reflux during ≈1 h to asuspension of magnesium tunings (2.43 g) and a trace iodine in diethylether (50 mL). When the addition was completed, the mixture was refluxedfor four hours, then cooled to 0° C. Dry THF (50 ml) was added followedby slow addition of a solution of cyclobutanone (7.01 g, 100 mmol) indiethyl ether (50 mL) and the mixture was left to attain rt. Afterstirring for two h, a cool saturated solution of NH₄Cl (500 ml) wasadded and the mixture was stirred for 15 minutes, then extracted twicewith EtOAc. The organic phase was washed with brine, dried with sodiumsulfate and evaporated under reduced pressure. The product was purifiedby column chromatography on silica gel, which gave the title compound(12.5 g, 42%).

Step b) 4-cyclobutylphenol (Phenol 7)

Pd 10% on carbon (2.55 g, 21.5 mmol) was added under argon to a solutionof Ph7-a (12.4 g, 41.4 mmol) in abs EtOH (110 mL) the and the mixturewas hydrogenated at 45 psi at rt for 18 h. The catalyst was filtered of,washed with ethanol and the solution was concentrated.

The product was purified by silica gel chromatography (isohexane-EtOAc).Appropriate fractions were pooled and concentrated and the residuecrystallized from petrol ether which gave the title compound (3.15 g,51%).

Phenol 8

4-(1-Methylcyclopentyl)phenol (Ph-8)

A solution of 1-methylcyclopentanol (2.00 g, 20.0 mmol) and phenol (2.07g, 22.0 mmol) in pentane (50 mL) were added dropwise during 30 min to asuspension of fresh AlCl₃ (1.33 g, 10 mmol) in pentane (100 mL). Theresulting mixture was stirred under N₂ at rt for 72 h, then the reactionmixture was poured into water/ice and HCl (12 M, 20 mmol, 1.66 mL). Theorganic phase was washed with water (50 mL) and brine (50 mL), dried(Na₂SO₄) filtered and concentrated. The crude was purified by columnchromatography on silica (MeOH-DCM), which gave the title compound (426mg, 12%).

Phenol 9

Step a) 2-(4-Bromo-3-methylphenoxy)tetrahydro-2H-pyran (Ph9-a)

pTs (16 mg, 0.086 mmol) was added to a solution of4-bromo-3-methylphenol (4.0 g, 21.4 mmol) in 3,4-dihydro-2-H-pyran (16mL, 175 mmol). The reaction mixture was stirred at room temperature for1 h, then diluted with diethyl ether and washed with 1M NaOH (aq) andwater. The organic phase was dried (Na₂SO₄) filtered and concentrated.The crude was purified by column chromatography on silica(EtOAc/heptane) which gave the title compound (3.32 g, 57%).

Step b) 2-(4-Cyclopropyl-3-methylphenoxy)tetrahydro-2H-pyran (Ph9-b)

Ph9-a (3.12 g, 11.5 mmol), ZnBr₂ (2.59 g, 11.5 mmol),tri-tert-butylphosphine tetrafluoroborate (0.2 g, 0.69 mmol) andPd(OAc)₂ (258 mg, 1.15 mmol) were put in a flask and the flask wasflushed with N₂ a couple of times. THF (10 mL) was added while stirring,followed by dropwise addition of 0.5 M cyclopropylmagnesium bromide inTHF (35 mL, 17.4 mmol) during 5 minutes. The mixture was stirred at rton, then filtered through a Celite plug, eluted with MeOH. The solutionwas concentrates and the crude was purified by column chromatography onsilica (EtOAc/heptane) which gave the title compound (1.69 g, 57%).

Step c) 4-Cyclopropyl-3-methylphenol (Phenol 9)

Ph9-b (1.70 g, 7.30 mmol) was dissolved in MeOH (20 ml) and pTsxH₂O (318mg, 1.67 mmol) was added. The mixture was stirred at 22° C. for 30minutes, then concentrated. The crude was purified by columnchromatography (EtOAc/heptane), which gave the title compound (704 mg,65%).

Phenol 10

Step a) 4-cyclopropyl-1-methoxy-2-methylbenzene (Ph10-a)

4-Bromo-1-methoxy-2-methylbenzene (4.39 g, 21.9 mmol) was reacted withcyclopropylmagnesium bromide according to the procedure described in Ph9step b, which gave the title compound (1.54 g, 43%).

Step b) 4-cyclopropyl-2-methylphenol (Phenol 10)

BBr₃ (5 mL, 5 mmol) was added under N₂ at 0° C. to a solution of Ph10-a(1.54 g, 9.49 mmol) in DCM (7.5 mL). The reaction was stirred for 2 h,then quenched with MeOH (3 mL) and concentrated. The crude was dissolvedin EtOAc and washed with brine. The organic phase was dried (Na₂SO₄),filtered and concentrated. The crude product was purified by columnchromatography on silica, which gave the title compound (826 mg, 59%).MS 147.11 [M−H]⁻.

Phenol 11

4-cyclopropyl-3-methoxyphenol (Phenol 11)

The title compound was prepared from 4-bromo-3-methoxyphenol (1.11 g,5.49 mmol) according to the procedure described for the preparation ofPhenol 9. Yield 40%.

Phenol 12

Step a) 3-(dimethylamino)-1-(3-hydroxyphenyl)propan-1-one (Ph12-a)

A few drops of HCl were added to a solution of 3-hydroxy acetophenone(4.08 g, 30 mmol), paraformaldehyde (4.05 g, 45 mmol) and dimethylaminehydrochloride (2.69 g, 33 mmol) in absolute EtOH (100 mL) and thereaction mixture refluxed for 18 h. Additional dimethylaminehydrochloride (0.55 eq., 1.22 g), paraformaldehyde (0.5 eq., 1.35 g) andHCl (0.5 mL) were added and the reaction mixture refluxed for additional4 h, then cooled to rt. The precipitated white solid was collected andwashed with cold EtOH (50 mL) and cold acetone (10 mL) and then freezedried, which gave the title compound (2.59 g, 38%) that was used in thenext step without further purification.

Step b) cyclopropyl(3-hydroxyphenyl)methanone (Phenol 12)

NaH (60% mineral oil dispersion) (1.13 g, 28.2 mmol) was added inportions at rt to a stirred suspension of trimethylsulfoxonium iodide(6.20 g, 28.2 mmol) in DMSO (100 mL). After 1 h, solid Ph12-a (2.59 g,11.3 mmol) was added in portions under stirring and cooling. Thereaction mixture was stirred at rt for 40 h, then poured into cold water(200 mL) and extracted with DCM (3×100 mL). The organic phase was washedwith a saturated aqueous solution of NH₄Cl (2×100 mL), dried (Na₂SO₄),filtered and concentrated. The afforded crude was purified by columnchromatography on silica (MeOH/DCM) which gave the title compound (883mg, 48%).

Phenol 13

Step a) cyclopropyl(4-hydroxyphenyl)methanone (Ph13)

p-Hydroxy-γ-chlorobutyrophenone (4.95 g) was added in portions duringapproximately 30 min to a solution of NaOH (8 mL, aq, 50% w/w), thenNaOH (35 mL, aq. 25% w/w) was added followed by p-hydroxyγ-chlorobutyrophenone (4.95 g) in one portion. The temperature waslowered to 140° C. and NaOH (8 g) was added. After 90 min, H₂O (10 ml)was added, and after additional 60 min, the reaction mixture was cooled,diluted with H₂O and neutralized with HOAc (≈27-30 ml) to pH≈7 Theformed precipitate was filtered, washed with H₂O and dried in vacuum.The solids were triturated in CHCl₃ (200 ml) at 40° C. during 10 min,then at RT overnight. The slurry was heated to 40° C. during 30 min,then filtered. The filtrate was dried (MgSO₄), filtered and concentratedto ≈70 ml. Hexane was added and an oil was formed that eventually becamecrystals. The slurry was filtered, solids washed with CHCl₃/hexane anddried, which gave the title compound (4.15 g, 51%).

Phenol 14

Step a) 3-(1-hydroxy-2,2-dimethylpropyl)phenol (Ph14-a)

t.Bu-MgBr (1.5 eq.) was added dropwise during 30 minutes to a cold (−10°C.) mixture of 3-hydroxybenzaldehyde (2.00 g, 16.4 mmol) in diethylether (20 mL). During the addition THF (20 mL) was added. The mixturewas allowed to reach 23° C. and stirred for 6 hours. More t.Bu-MgBr (0.7eq.) was added and the mixture was left stirring over night, then cooledand the reaction was quenched with aqueous saturated NH₄Cl. to give.EtOAc was added to the mixture followed by addition of 1 M aqueous HCluntil a homogeneous mixture was obtained. The phases were separated andthe organic phase was washed with brine, dried (Na₂SO₄), filtered andconcentrated. The afforded crude was purified by column chromatography,which gave the title compound (1.1 g, 37%).

Step b) 1-(3-hydroxyphenyl)-2,2-dimethylpropan-1-one (Ph14)

To an oven dried round bottomed flask was added 3 Å MS and pyridiniumchlorochromate (PCC) (1.97 g, 9.15 mmol) followed by dry DCM (5 mL). Themixture was stirred at 20° C. for 5 minutes whereafter a mixture ofAA8019 (1.10 g, 6.10 mmol) in DCM (5 mL) was added slowly. Aftercomplete oxidation the mixture was filtered through a pad of Celite,washing the pad with diethyl ether. The filtrate was concentrated. Thecrude was purified by column chromatography which gave the titlecompound (402 mg, 37%). MS 179.25 [M+H]+.

Phenol 15

1-(4-Hydroxyphenyl)-2,2-dimethylpropan-1-one (Ph15)

4-hydroxybenzaldehyde (3 g, 24.6 mmol) was reacted according to theprocedure described for the preparation of Phenol 14, which gave thetitle compound (538 mg, 17%).

Amino Acid 1

Step a) (S)-(S)-Sec-butyl 2-((tert-butoxycarbonyl)amino)propanoate(AA1-a)

L-Boc-Alanine (2.18 g, 11.5 mmol) was dissolved in dry DCM (40 mL) andthe alcohol (R)-butan-2-ol (938 mg, 12.6 mmol) was added. The mixturewas cooled to about 5° C. and EDC (3.31 g, 17.2 mmol) was added in oneportion followed by portionwise addition of DMAP (140 mg, 1.15 mmol).The mixture was allowed to attain room temperature and stirredovernight, then diluted with ethyl acetate (˜300 ml) and the organicphase was washed three times with a saturated solution of sodiumhydrogen carbonate and once with brine. The organic phase was dried oversodium sulfate and concentrated under reduced pressure. The product wasisolated by silica gel chromatography eluted with isohexane and 10%ethyl acetate, which gave the title compound (2.78 g, 98%).

Step b) (S)-(S)-Sec-butyl 2-aminopropanoate (AA1-b)

A mixture of AA1-a (2.77 g, 11.3 mmol) and p-toluene sulfonic acid monohydrate (2.15 g, 11.3 mmol) in EtOAc (45 mL) was stirred for 16 h at 65°C., then concentrated under reduced pressure. The afforded residue wascrystallised from diethyl ether, which gave the title compound (3.20 g,89%).

Amino Acid 2

(S)-(R)-Pentan-2-yl 2-aminopropanoate (AA2)

The procedure described for the preparation of AA1 was followed butusing (R)-pentan-2-ol instead of (R)-butan-2-ol, which gave the titlecompound (4.6 g).

Amino Acid 3

(S)-(S)-Pentan-2-yl 2-aminopropanoate (AA3)

The procedure described for the preparation of AA1 was followed butusing (S)-pentan-2-ol instead of (R)-butan-2-ol, which gave the titlecompound (8.3 g).

The following intermediates were prepared and can be used in thepreparation of compounds of the invention:

INTERMEDIATE 1

Step a) (R)-4-Fluorobenzyl 2-((tert-butoxycarbonyl)amino)propanoate(I-1a)

Boc-L-AlaOH (19.92 mmol), DMAP (1.99 mmol) and (4-fluorophenyl)methanol(23.9 mmol) were dissolved in CH₂Cl₂ (100 mL). To this solution wasadded triethylamine (23.9 mmol) followed by EDC (23.9 mmol) and theresulting reaction mixture was stirred overnight at room temperatureunder N₂. The reaction mixture was diluted with CH₂Cl₂ (100 mL), washedwith saturated aqueous solution of NaHCO₃ (2×50 mL), saturated aqueoussolution of NaCl (2×50 mL), dried (Na₂SO₄) and concentrated. Theafforded residue was purified by column chromatography on silica geleluted with n-hexane-EtOAc (95:5 to 60:40) which gave the title compound(4.44 g) as a white waxy solid. MS: 296 [M−H]⁻.

Step b) (R)-4-fluorobenzyl 2-aminopropanoate (I-1b)

Compound I-1a (14.93 mmol) was dissolved in 4M HCl/dioxane (40 mL) andstirred at room temperature for 30 minutes and evaporated to drynesswhich gave the hydrochloride salt of the title compound (3.4 g) as awhite powder. MS: 198 [M+H]⁺.

Step c) (2R)-4-fluorobenzyl2-((chloro(phenoxy)phosphoryl)amino)propanoate (I-1)

PhOPOCl₂ (4.28 mmol) was added dropwise at −78° C. to a solution ofcompound I-5b (4.28 mmol) in CH₂Cl₂. followed by dropwise addition oftriethylamine (8.56 mmol). The resulting reaction mixture was stirred at−78° C. under Ar and allowed to attain room temperature overnight. Thereaction mixture was evaporated on silica gel and purified bychromatography (n-hexane/EtOAc (88:12)-(0:100)). which gave the titlecompound (769 mg) as a mixture of stereoisomers at the phosphorus atom.³¹P-NMR (CDCl₃) δ: 7.85 (s) and 7.54 (s).

INTERMEDIATE 2

Step a) (S)-(R)-Sec-butyl 2-((tert-butoxycarbonyl)amino)propanoate(I-2a)

L-Boc-Alanine (2.18 g, 11.5 mmol) was dissolved in dry DCM (40 mL) andthe alcohol (R)-butan-2-ol (938 mg, 12.6 mmol) was added. The mixturewas cooled to about 5° C. and EDC (3.31 g, 17.2 mmol) was added in oneportion followed by portionwise addition of DMAP (140 mg, 1.15 mmol).The mixture was allowed to attain room temperature and stirredovernight, then diluted with ethyl acetate (˜300 ml) and the organicphase was washed three times with a saturated solution of sodiumhydrogen carbonate and once with brine. The organic phase was dried oversodium sulfate and concentrated under reduced pressure. The product wasisolated by silica gel chromatography eluted with isohexane and 10%ethyl acetate, which gave the title compound (2.78 g, 98%).

Step b) (S)-(R)-Sec-butyl 2-aminopropanoate (I-2b)

A mixture of I-10a (2.77 g, 11.3 mmol) and p-toluene sulfonic acid monohydrate (2.15 g, 11.3 mmol) in EtOAc (45 mL) was stirred for 16 h at 65°C., then concentrated under reduced pressure. The afforded residue wascrystallised from diethyl ether, which gave the title compound (3.20 g,89%).

Step c) (2S)-(R)-Sec-butyl2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-2)

Phenyl dichlorophosphate (1 eq) was added under nitrogen at −30° C. to asolution of Compound I-10b (3.15 g, 9.92 mmol) in DCM (75 ml), followedby dropwise addition of triethylamine (2 eq). The mixture was allowed toattain room temperature and stirred overnight, then cooled to about 5°C. and 4-nitrophenol (1 eq, 15 mmol) was added as a solid followed bydropwise addition of triethylamine (1 eq g, 15 mmol) and the mixture wasstirred for 4 hours at room temperature, then concentrated under reducedpressure, diluted with ethyl acetate (40 ml) and ether (40 ml) and leftat room temperature overnight. The triethylamine-HCl salt was filteredof and the filtrate was concentrated under reduced pressure. Theafforded residue was purified by column chromatography on silica geleluted with iso-hexane-ethyl acetate, which gave the title compound(4.19 g, 79%) as a mixture of stereoisomers at the phosphorus atom.

The following compounds were prepared as mixtures of stereoisomers atthe phosphorus atom according to the procedure described for thepreparation of I-2 using the appropriate alcohol:

I-# Structure alcohol I-3

cyclopropylethanol I-4

cyclopentylmethanol I-5

pentan-3-ol I-6

2-propylpentan-1-ol

INTERMEDIATE 7

Step a) (S)-cyclooctyl 2-aminopropanoate (I-7a)

p-Toluenesulfonic acid monohydrate (3.6 g, 19.1 mmol) was added to aslurry of L-alanine (1.7 g, 19.1 mmol) and cyclooctanol (25 ml, 191mmol) in toluene (100 ml). The reaction mixture was heated at refluxtemperature for 25 h and water was removed from the reaction using aDean-Stark trap. The mixture was concentrated under reduced pressure andthe residue kept under vacuum over night. To the residue (27 g) wasadded diethyl ether (100 ml). The white precipitate was collected byfiltration, washed with diethyl ether (3×50 ml) and dried under vacuumwhich gave the title compound (4.84 g, 68%).

Step b) (2S)-cyclooctyl2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-7)

Compound I-7a was reacted according to the method described for thepreparation of I-2 step c, which gave the title compound (4.7 g, 76%) asa mixture of stereoisomers at the phosphorus atom.

INTERMEDIATE 8

(2S)-cycloheptyl2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-22)

The procedure described for the preparation of compound I-7 was followedbut using cycloheptanol (27 ml, 224 mmol) instead of cyclooctanol, whichgave the title compound (5.72 g, 55%) as a mixture of stereoisomers atthe phosphorus atom.

INTERMEDIATE 9

(2S)-Cyclohexyl 2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate(I-23)

The procedure described for the preparation of I-2 step c was followedbut using (S)-cyclohexyl 2-aminopropanoate instead of(S)-3,3-dimethylbutyl 2-aminopropanoate, which gave the title compound(10.6 g, 82%) as a mixture of stereoisomers at the phosphorus atom.

INTERMEDIATE 10

(S)-2-Ethylbutyl 2-((bis(4-nitrophenoxy)phosphoryl)amino)propanoate(I-10)

(S)-2-Ethylbutyl 2-aminopropanoate (5 g, 14.49 mmol) was added to asolution of bis(4-nitrophenyl) phosphorochloridate (6.14 g, 17.1 mmol)in DCM (50 ml), the mixture was cooled in an ice bath and Et₃N (4.77 mL,34.2 mmol) was added drop wise. The cooling was removed after 15 min andthe reaction mixture was stirred at 23° C. until complete reactionaccording to TLC. Diethyl ether was then added, the mixture was filteredand the filtrate was concentrated and purified by column chromatographyon silica which gave the title compound (2.05 g, 82%).

INTERMEDIATE 11

Step a) (S)-isopropyl 2-aminopropanoate (I-11a)

SOCl₂ (29 mL, 400 mmol) was added dropwise at 0° C. to a suspension ofthe HCl salt of L-alanine (17.8 g, 200 mmol) in isopropanol (700 mL).The suspension was stirred at room temperature over night, thenconcentrated, which gave the title compound (29.2 g, 87%).

Step b) (2S)-Isopropyl2-(((((S)-1-isopropoxy-1-oxopropan-2-yl)amino)(4-nitrophenoxy)phosphoryl)-amino)propanoate(I-11)

A solution of 4-nitrophenyl dichlorophosphate (1.8 g 7 mmol) in DCM wasadded dropwise at −60° C. to a solution of the amine I-11a (2.35 g, 14mmol) and triethylamine (7.7 mL, 56 mmol) in DCM. The reaction mixturewas allowed to attain room temperature, stirred over night, concentratedand then diluted with ethyl acetate and ether and left at roomtemperature overnight. The triethylamine-HCl salt was filtered of, thefiltrate was concentrated under reduced pressure and the affordedresidue was purified by chromatography on silica gel eluted withiso-hexane-ethyl acetate, which gave the title compound (1.6 g, 50%).

INTERMEDIATE 12

Step a) (S)-Neopentyl 2-((tert-butoxycarbonyl)amino)propanoate (I-12a)

EDAC and DMAP was added in portions at −5° C. to a solution ofBoc-alanine (18.9 g, 100 mmol) and neopentylalcohol (13.0 mL, 120 mmol)in DCM (200 mL). The reaction mixture was allowed to attain roomtemperature and stirred for 72 h. EtOAc (700 mL) was added and theorganic phase was washed three times with a saturated solution of NaHCO₃and once with brine, then concentrated. The afforded residue waspurified by column chromatography eluted with hexane-EtOAc 90/10 to80/20, which gave the title compound (21 g, 81%).

Step b) (S)-Neopentyl 2-aminopropanoate (I-12b)

p-Toluene sulfonic acid (15.6 g, 82.0 mmol) was added at −65° C. to asolution of the Boc protected amine I-12a (21.1 g, 82.0 mmol) in EtOAc(330 mL). The reaction mixture was stirred at −65° C. for 8 h. then leftto attain room temperature overnight. The mixture was then filtered andconcentrated which gave the title compound (21 g, 78%).

(2S)-Neopentyl2-(((((S)-1-(neopentyloxy)-1-oxopropan-2-yl)amino)(4-nitrophenoxy)-phosphoryl)amino)propanoate(I-12)

4-Nitrophenol dichlorophosphate was added dropwise during 1 h at −50° C.to a solution of the amine I-12b (3.90 g, 24.5 mmol) in DCM (100 mL).The reaction mixture was allowed to attain room temperature, stirredovernight, concentrated and then diluted with diethyl ether and left atroom temperature overnight. The mixture was filtered, the filtrate wasconcentrated under reduced pressure and the afforded residue waspurified by chromatography on silica gel eluted with iso-hexane-ethylacetate, which gave the title compound (4.8 g, 77%).

INTERMEDIATE 13

(2S)-Ethyl 2-((chloro(phenoxy)phosphorothioyl)amino)propanoate (I-13)

Thiophosphoryl chloride (0.27 mL, 2.62 mmol) was added at −35° C. underN₂ to a solution of phenol (247 mg, 2.62 mmol) in a mixture of dry DCM(8.8 mL) and dry THF (4.4 mL). After 5 min, triethylamine (365 μL, 2.62mmol) was added dropwise and the reaction mixture was stirred at −35° C.for 3 h. Alanine ethyl ester×HCl (403 mg, 2.62 mmol) was added and thereaction mixture was stirred for 5 min at −35° C. whereaftertriethylamine (731 μL, 5.24 mmol) was added dropwise. The temperaturewas slowly allowed to reach rt overnight (17h). The reaction mixture wasdiluted with Et₂O, filtered and concentrated under reduced pressure.Flash chromatography (hexane:EtOAc 8:1) of the afforded crude productgave the title compound (659 mg, 82%) as a mixture of stereoisomers atthe phosphorus atom as a clear oil. MS 306.18 [M−H]⁻.

INTERMEDIATE 14

(2S)-Neopentyl2-((chloro(4-chlorophenoxy)phosphorothioyl)amino)propanoate (I-14)

4-Chlorophenol (381 μL, 3.87 mmol) was added under nitrogen in one to asolution at −30° C. of thiophosphoryl chloride (400 μL, 3.87 mmol) inDCM followed by dropwise addition of triethylamine (1.62 mL, 11.6 mmol).The reaction was stirred for 2 h while the temperature was allowed toreached +5° C. The pTs salt of (S)-neopentyl 2-aminopropanoate (1.28 g,3.87 mmol) was added and the mixture was cooled to −30° C. Triethylamine(1.62 L, 11.6 mmol) was added dropwise and the reaction allowed to reachroom temperature and stirred over the weekend. The mixture wasconcentrated onto silica-gel and the residue purified by flashchromatography using hexanes/ethyl acetate: 7/1 which gave the titlecompound as a mixture of stereoisomers at the phosphorus atom (807 mg,54%). MS 368.34 [M+H]⁺.

INTERMEDIATE 15

(2S)-methyl2-((chloro(naphthalen-1-yloxy)phosphorothioyl)amino)propanoate (I-15)

Thiophosphoryl chloride (1 eq) was added at −35° C. under N₂ to asolution of naphthol (1 eq) in a mixture of dry DCM (10 mL) and dry THF(5 mL). After 5 min, triethylamine (1 eq) was added dropwise and thereaction mixture was stirred at −35° C. for 3 h. (S)-methyl2-aminopropanoate (1 eq) was added and the reaction mixture was stirredfor 5 min at −35° C. whereafter triethylamine (2 eq) was added dropwise.The temperature was slowly allowed to reach rt overnight. The reactionmixture was diluted with Et₂O, filtered and concentrated under reducedpressure. Flash chromatography (hexane:EtOAc 8:1) of the afforded crudeproduct gave the title compound as a mixture of stereoisomers at thephosphorus atom. Yield 8.0% MS 564.24 [M+H]⁺.

The following intermediates were prepared as a mixture of stereoisomersat the phosphorus atom according to the method described forIntermediate 13 using the appropriate phenol and amino acid ester.

I-# R₁₆ R_(meta) R_(para) 16 (2S)-2-ethylbutyl cyclopropyl H 17 ethylcyclopropyl H 18 (S)-2-pentyl cyclopropyl H 19 isopropyl H cyclopropyl20 ethyl H cyclopropyl 21 methyl H cyclopropyl 77 isobutyl H cyclopropyl23 isopropyl H H 24 methyl methylcyclopropyl H 25 isopropyl cyclopropylH 26 isobutyl cyclopropyl H 27 n-butyl cyclopropyl H 28 methylcyclopropyl H 29 isopropyl cyclobutyl H 30 methyl H H 31 isopropyl H H

INTERMEDIATE 32

(2S)-(R)-sec-butyl2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-32)

Et₃N (10.9 mL, 78.1 mmol) was added dropwise at −70° C. under nitrogenduring 15 minutes to a stirred solution of the pTs salt of(S)-(R)-sec-butyl 2-aminopropanoate (12.0 g, 37.7 mmol) in DCM (50 mL).To this mixture was added a solution of phenyl dichlorophosphate (5.61mL, 37.7 mmol) in DCM (50 mL) during 1 h. The reaction mixture wasstirred at −70° C. for additional 30 minutes, then allowed to warm to 0°C. during 2 h and stirred for 1 h. A solution of pentafluorophenol (6.94g, 37.7 mmol) and Et₃N (5.73 mL, 41.1 mmol) in DCM (30 mL) was added tothe mixture during 20 minutes. The crude mixture was allowed to stir at0° C. for 18 h, and was then concentrated. The residue was taken in THF(100 mL), insolubles were filtered off and washed several times withTHF. The solvent was evaporated and the residue triturated withtert-butyl methyl ether. Insolubles were filtered off and washed withtert-butyl methyl ether. The combined filtrate was concentrated and thecrude solid sonicated with n-hexane/EtOAc (80:20; 100 mL). The solid wasfiltered, washed with n-hexane/EtOAc (80:20) which gave the pureP-stereoisomer of the title compound as a white solid (2.3 g, 13%).

INTERMEDIATE 33

(2S)-ethyl 2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate(I-33)

The title compound was prepared as a single stereoisomer at thephosphorus atom according to the method described for I-32, but startingfrom the HCl salt of (S)-ethyl 2-aminopropanoate (11.0 g, 71.1 mmol).Yield 8.56 g, 27%.

INTERMEDIATE 34

(2S)-2-ethylbutyl2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-34)

The title compound was prepared as a single stereoisomer at thephosphorus atom according to the method described for I-32, but startingfrom the pTs salt of (S)-2-ethylbutyl 2-aminopropanoate (18.8 g, 54.4mmol). Yield 27.0 g, 99%.

LC-MS 496.44 [M+H]⁺.

INTERMEDIATE 35

(2S)-butyl 2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate(I-35)

Phenyl dichlorophosphate (12.4 mL, 83.1 mmol) was added to a cooled(−20° C.) slurry of (S)-butyl 2-aminopropanoate (26.4 g, 83.1 mmol) inDCM (200 mL). The mixture was stirred for 10 min then Et₃N (25.5 mL, 183mmol) was added dropwise for 15 min. The mixture was stirred at −20° C.for 1 h then at 0° C. for 30 min. The mixture was kept cooled in anice-bath and pentafluorophenol (15.3 g, 0.08 mol) was added followed bya dropwise addition of Et₃N (11.6 mL, 0.08 mol). The mixture was stirredover night and slowly taken to 20° C. Diethyl ether was added and themixture was filtered through Celite, concentrated and purified by columnchromatography on silica gel eluted with petroleum ether/EtOAc(9:1->8:2). Appropriate fractions were pooled, concentrated andcrystallized from petroleum ether/EtOAc which gave the pureP-stereoisomer of the title compound as a white solid (2.23 g, 5.8%).

INTERMEDIATE 36

(2S)-Cyclohexyl2-(((perfluorophenoxy(phenoxy)phosphoryl)amino)propanoate (I-36)

Phenyl dichlorophosphate (11.1 mL, 74.4 mmol) was added in one portionat −15° C. to a solution of L-alanine cyclohexyl ester (25.5 g, 74.4mmol) in DCM (250 mL). The resulting mixture was stirred for 10 min,then triethylamine (2.2 eq.) was added over a period of 10 min and thereaction was allowed to proceed cold for 30 min at −15° C. and then atroom temperature for 72 h. The reaction was cooled on ice andpentafluorophenol (13.7 g, 74.4 mmol) was added, followed by addition oftriethylamine (1 eq.) over 10 min. The reaction was allowed to attain rtand was stirred for 30 min. Insoluble material was filtered off througha pad of Celite and the filter cake was washed with DCM (100 mL). Thesolvent was evaporated and the residue dried in vacuum, then taken intoEtOAc (200 mL) and stirred for 20 min. Insoluble material was filteredoff through a pad of Celite and the cake washed with EtOAc (75 mL) andthe solution was left at 5° C. overnight. The formed crystals weredissolved in EtOAc and the solution was washed with 2 M NaOH (×1), 2 MHCl (×1) dried (Na₂SO₄) and concentrated, which gave (2.37 g, 6%) almostpure diastereoisomer of the title compound (de=˜90%).

INTERMEDIATE 37

(2S)-Isopropyl2-(((benzo[d][1,3]dioxol-5-yloxy)(perfluorophenoxy)phosphoryl)amino)propanoate(I-37)

POCl₃ (1.79 ml, 19.2 mmol) was added under N₂ at −78° C. to a solutionof sesamol (2.65 g, 19.2 mmol) in DCM (60 mL), followed by drop wiseaddition of Et₃N (2.67 ml, 19.2 mmol). The mixture was stirred for 4 hat −20 to −30° C. The mixture was cooled to −78° C. and a solution of(S)-isopropyl 2-aminopropanoate (3.22 g, 19.2 mmol) in DCM (10 mL) wasadded dropwise, followed by addition of Et₃N (5.62 ml, 40.3 mmol) over15 min. The reaction mixture was allowed to attain rt and stirred overnight. The temperature of the reaction mixture was then lowered to 0° C.and pentafluorophenol (3.53 g, 19.2 mmol) was added in one portionfollowed by dropwise addition of Et₃N (2.67 ml, 19.2 mmol). The obtainedslurry was stirred at 0° C. When the reaction was completed as judged byLC-MS, the mixture was filtered and the solid was washed with cold DCM.The filtrate was concentrated and redissolved in tert-butyl ether,filtered again and then concentrated. EtOAc:Hexane 20:80 was added andthe obtained slurry heated gently until a clear solution was obtained.The solution allowed to reach rt and then put at −20° C. After 1 hourcrystals was formed, filtered off, washed several times with hexane andthen dried under vacuum, yield: 1.8 g. The mother liquid wasconcentrated and the crystals formed filtered off and dried undervacuum, yield: 5.5 g. Total yield: 7.3 g, 69% of the title compound as asingle stereoisomer at the phosphorus atom. MS ES+ 498.06 [M+H]⁺.

The following intermediates were prepared as single stereoisomers at thephosphorus atom according to the method described for Intermediate 37using the appropriate phenol and amino acid ester.

I-# R_(ortho) R_(meta) R_(para) Yield MS 38   methoxy H H 62% na 39   HH methoxy 63% na 40¹ H cyclopropyl H 27% 494.2 [M + H]⁺ 43   Hcyclobutyl H 20% 508.0 [M + H]⁺ 44¹ H 1- H 11% 508.0 methylcyclopropyl[M + H]⁺ 45¹ H H 1- 41% 506.5 methylcyclopropyl [M − H]⁻¹Pentafluorophenol was added at −78° C. not at 0° C. as in I-37

INTERMEDIATE 41

(2S)-(S)-Sec-butyl2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-41)

The title compound was prepared as a single stereoisomer at thephosphorus atom according to the method described for I-32, but startingfrom (S)-(S)-sec-butyl 2-aminopropanoate (12.0 g, 37.8 mmol) instead of(S)-(R)-sec-butyl 2-aminopropanoate. Yield: 3.33 g, 19%.

INTERMEDIATE 42

(2S)-Propyl 2-((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate(I-42)

The title compound was prepared as a single stereoisomer at thephosphorus atom according to the method described for I-35, but startingfrom the HCl salt of (S)-propyl 2-aminopropanoate (5.62 g, 33.53 mmol))instead of the pTs salt (S)-(R)-sec-butyl 2-aminopropanoate. The productwas recrystallized from isopropyl ether. Yield: 5.8 g (38%). LC-MS ES+454.1 [M+H]⁺.

INTERMEDIATE 46

Step a) (S)-(R)-1-Methoxypropan-2-yl2-((tert-butoxycarbonyl)amino)propanoate (I-46a)

EDC (6.08 g, 0.03 mol) and 4-(dimethylamino)pyridine (0.48 g, 0.004 mol)were added to a solution of Boc-L-alanine (5 g, 0.03 mol) and(R)-(−)-1-methoxy-2-propanol (2.59 ml, 0.03 mol) at 0° C. The reactionmixture was left stirring on a melting ice-water bath and was thenstirred at room temperature for 72 h.

The reaction mixture was concentrated to −½ the volume, diluted withethyl acetate (400 mL) and washed with saturated aqueous NH₄Cl (200 ml),10% aqueous citric acid (50 mL) and saturated aqueous NaHCO₃ (200 mL).The organic layer was dried (Na₂SO₄), filtered and concentrated.

The crude product was purified by silica gel column chromatography(Biotage SNAP ultra 100 g, gradient of 5-30% ethyl acetate in heptane)which gave the title compound as a clear oil (5.90 g, 85%).

Step b) (S)-(R)-1-methoxypropan-2-yl 2-aminopropanoate (I-46b)

A solution of I-46a (5.88 g) in 4M HCl in dioxane (50 mL) was stirredfor 90 min, then concentrated and the residue freeze dried from dioxane(25 mL), which gave the title compound as the hydrochloride (5.19 g, 99%o).

Step c) (2S)-(R)-1-Methoxypropan-2-yl2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)-propanoate (I-46)

Triethylamine (9.25 mL, 66.4 mmol) was added dropwise to a cooled (0°C.) solution of (S)-(R)-1-methoxypropan-2-yl 2-aminopropanoatehydrochloride (5.18 g, 22.1 mmol) in DCM (35 mL). The mixture was cooledto −78° C. and a solution of phenyl dichlorophosphate (3.29 mL, 22.1mmol) in DCM (20 mL) was added. The mixture was stirred for 10 min thenEt₃N (25.5 mL, 183 mmol) was added dropwise for 15 min. The mixture wasstirred at −78° C. for 5 min then at 0° C. for 2 h. Pentafluorophenol(4.07 g, 22.1 mmol) and Et₃N (3.39 mL, 23.3 mmol) in DCM (20 mL) wasadded dropwise then the reaction mixture was slowly left to attain roomtemperature and was stirred over night. The mixture was concentrated andTHF (50 mL) was added. Solids filtered off and washed with THF (3×25 mL)The filtrate was concentrated and the residue dissolved in tert-butylmethyl ether (50 ml) with help of sonification. Heptane (50 ml) wasadded and the product precipitated from the solution upon standing atroom temperature for 1 h. More heptane was added (50 ml) and the solidwas removed by filtration. The precipitate was washed with tert-butylmethyl ether/heptane 1:2 (50 ml) and heptane (50 ml). The precipitatewas dried under vacuum which gave the title compound as pure isomeraccording to NMR. (4.32 g, 40%). LC-MS ES+ 484.34 [M+H]⁺.

INTERMEDIATE 47

Step a) (S)-1,3-Dimethoxypropan-2-yl2-((tert-butoxycarbonyl)amino)propanoate (I-47-a)

EDC (2.79 g, 14.5 mmol), crystalline 4-(dimethylamino)pyridine (229 mg,1.88 mmol) and Et₃N (5.27 ml, 37.8 mmol) were added to a solution ofBoc-L-alanine (2.42 g, 12.8 mmol) and 1,3-dimethoxypropan-2-ol (1.52 g,12.6 mmol). The reaction mixture was stirred at room temperature for 72h. then diluted with EtOAc and washed with, NaHCO₃ (aq, ×2), 0.1M HCl(aq, ×2), dried (Na₂SO₄) and concentrated. The afforded crude productwas used as is in the next step.

Step b) (2S)-1,3-Dimethoxypropan-2-yl2-(((perfluorophenoxy)(phenoxy)phosphoryl)-amino)propanoate (I-47)

I-47a (3 g, 10.8 mmol) was stirred in 4M HCl in THF (15 mL, 60 mmol) at22° C. for 2 h then concentrated and co-evaporated twice with toluene.The afforded oil that was dissolved in DCM (40 ml) and phenyldichlorophosphate (1.62 mL, 10.8 mmol) was added. The mixture was cooledon an ice bath and after 15 min Et₃N (3.32 mL, 23.8 mmol) was addedslowly. The mixture was stirred at 4° C. for 18 h, then slowly taken to22° C. The mixture was again cooled to 0° C. and pentafluorophenol (2.01g, 10.9 mmol) was added followed by a dropwise addition of Et₃N (1.51mL, 10.8 mmol). The mixture was stirred at 0° C. for 1 h then at 22° C.for 5 h. The mixture was filtered, and the solids washed with EtOAc×3(total 150 mL). The combined organic phases were washed with NaHCO₃ (aq,×2) and brine, then dried (Na₂SO₄). The solution was put through a shortsilica column eluted with p.ether/EtOAc (8:2) appropriate fractions werecollected and concentrated and the afforded oil dissolved in diisopropylether and treated with heptane to give a light cloudy solution thatsolidified on standing. The mixture was left at 4° C. for 72 h. then thesolids were collected by filtration which gave the title compound as asingle stereoisomer at the phosphorus atom (333 mg, 6%). LC ES+ 514.0[M+H]⁺.

INTERMEDIATE 48

(2S)-Pentan-3-yl2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-48)

The title compound was prepared as a single stereoisomer at thephosphorus atom according to the method described for I-32, but startingfrom the HCl salt of (S)-pentan-3-yl 2-aminopropanoate (3.25 g, 16.6mmol)) instead of the pTs salt (S)-(R)-sec-butyl 2-aminopropanoate.Yield:8.0 g (18%). LC-MS ES+ 482.4 [M+H]⁺.

INTERMEDIATE 49

The title compound was prepared as a single stereoisomer at thephosphorus atom according to the procedure described in WO 2014/078427.

INTERMEDIATE 50

(2S)-Isopropyl2-(((perfluorophenoxy)(quinolin-6-yloxy)phosphoryl)amino)propanoate(I-50)

Phosphorus oxychloride (1.5 mL, 16.4 mmol) was added to DCM (40 ml) andthe mixture was cooled in a dry-ice/EtOH bath. 6-Hydroxyquinoline (2.38g, 16.4 mmol) was added followed by drop wise addition of Et₃N (2.28 mL,16.4 mmol) in DCM (5 mL). The mixture was stirred with cooling for 3 hthen isopropyl alanine (2.75 g, 16.4 mmol) was added followed by dropwise addition of Et₃N (4.57 ml, 32.8 mmol). The mixture was stirred withcooling for 5 h. Pentafluorophenol (3.02 g, 16.4 mmol) was addedfollowed by Et₃N (2.28 ml, 16.4 mmol) and the mixture was stirred for 72h. The mixture was diluted with EtOAc (200 mL) and washed with 0.1 M HCl(aq)×2, dried (Na₂SO₄) and concentrated. The residue was purified bysilica using petroleum ether/EtOAc (1:1) to give a beige solution thatsolidified in EtOAc/p-Ether. The solid was collected by filtration whichgave the title compound as a single stereoisomer at the phosphorus atom(787 mg, 9.5%).

INTERMEDIATE 51

(2S)-(S)-1-Methoxypropan-2-yl2-(((perfluorophenoxy(phenoxy)phosphoryl)amino)propanoate (I-51)

The title compound was prepared as a single isomer at the phosphorusatom according to the method described for I-46, but starting from(S)-(+)-1-methoxy-2-propanol (0.87 mL, 8.89 mmol) instead of(R)-(−)-1-methoxy-2-propanol. Yield: 604 mg, 14%. LC-MS (ES−) 481.5[M−H]⁻.

INTERMEDIATE 52 (S)-Isopropyl2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-52)

Step a) L-Alanine Isopropylester Hydrochloride

Thionylchloride (80.2 g, 0.674 mol, 1.5 eq) was added with cooling to2-propanol (400 mL) at −7 to 0° C. over a period of 30 minutes, followedby addition of L-alanine (40.0 g, 0.449 mol) at 0° C. A flow indicatorand a scrubber with a mixture of 27.65% sodium hydroxide (228 g) andwater (225 g) were attached to the outlet. The reaction mixture wasstirred at 67° C. for two hours, then at 70° C. for one hour and at20-25° C. over night. The reaction mixture was distilled at 47-50° C.under reduced pressure (250-50 mBar) from a 60° C. bath. When thedistillation became very slow, toluene (100 mL) was added to theresidual oil, and the distillation at 48-51° C. under reduced pressure(150-50 mBar) from a 60° C. bath was continued until it became veryslow. t-butylmethylether (tBME) (400 mL) was added to the residual oil,and the two-phase system was seeded under efficient stirring at 34-35°C. When crystallization was observed the mixture was cooled to 23° C.over a period of one hour, and the precipitate isolated by filtration.The filter cake was washed with tBME (100 mL) and dried to constantweight under reduced pressure without heating, which gave the titlecompound (67.7 g, 90%) as white solids.

Step b) (S)-Isopropyl2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-52)

Phenyl dichlorophosphate (62.88 g, 0.298 mol, 1.0 eq) was added undernitrogen to a solution of L-alanine isopropylester hydrochloride (50.0g, 0.298 mol) in DCM (310 mL) at 0° C.—the addition was completed bywash with DCM (39 mL). The mixture was cooled and triethylamine (63.35g, 0.626 mol, 2.1 eq) was added over a period of 70 minutes with coolingkeeping the temperature not higher than −14° C., the addition wascompleted by wash with DCM (39 mL). The mixture was stirred for one hourat −15 to −20° C., then heated to −8° C. and a solution ofpentafluorophenol (60.38 g, 0.328 mol, 1.1 eq) and triethylamine (33.19g, 0.328 mol, 1.1 eq) in DCM (78 mL) was added over a period of 42minutes with cooling keeping the temperature not higher than 0° C. - theaddition was completed by wash with DCM (39 mL). The mixture was stirredfor one hour at 0° C. and then over night at +5° C. The formedprecipitate was removed by filtration, and the filter cake washed withDCM (95 mL). The combined filtrates were washed at 5° C. with water(2×190 mL). The organic phase was distilled at 32-38° C. at reducedpressure (650-600 mBar), and distillation was continued until a residualvolume of approx. 170 mL partly crystallized mass was obtained. Ethylacetate (385 mL) was added, and the resulting clear solution wasdistilled at 43-45° C. under reduced pressure (300-250 mBar).Distillation was continued until a residual volume of approx. 345 mL wasobtained. The clear solution was cooled to 36° C., and crystallizationis induced by addition of seed crystals of (S)-isopropyl2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (20 mg)prepared as described in J. Org. Chem., 2011, 76, 8311-8319. The mixturewas cooled to 27° C. over a period of one hour, then n-heptane (770 mL)was added over a period of 47 minutes, and the mixture was stirred foran additional period of 37 minutes. Triethylamine (6.03 g, 0.2 eq) wasadded, and the mixture was stirred at 23-25° C. over night. Theprecipitate was isolated by filtration. The filter cake was washed withethyl acetate:n-heptane (1:9, 80 mL) and dried to constant under reducedpressure (below 0.1 mBar) without heating, which gave the title compound(75.64 g, 56%) as a single stereoisomer at the phosphorus atom as awhite crystalline material.

¹H NMR (CDCl₃, 300 MHz) δ 7.38-7.32 (m, 2 H), 7.27-7.24 (m, 2 H),7.23-7.19 (m, 1 H), 5.10-4.98 (m, 1 H), 4.20-4.08 (m, 1 H), 4.03-3.96(m, 1 H), 1.46 (dd, 7.2, 0.6 Hz, 3 H), 1.26-1.23 (2×d, 6 H);

¹³CNMR (CDCl₃, 100 MHz) δ 172.7 (d, J=8.8 Hz), 150.4 (d, J=7.1 Hz),143.4-143.0 (m), 141.0-140.2 (m), 140.0-139.8 (m), 137.6-137.2 (m),136.8-136.2 (m), 130.0 (d, J=0.82 Hz), 125.8 (d, J=1.4 Hz), 120.3 (d,J=5.0 Hz), 69.8, 50.6, (d, J=1.9 Hz), 21.8 (d, J=1.9 Hz), 21.2 (d, J=4.4Hz);

The crystallization properties and NMR spectral data of the titlecompound were in agreement with published data (J. Org. Chem., 2011, 76,8311-8319), thus confirming the S stereochemistry of the phosphorus atomof the title compound.

EXAMPLE 1

Step a)(4S,5R)-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)dihydrofuran-2(3H)-one(1a)

TIPS-chloride (16.4 g, 85 mmol) was added drop wise to an ice cooledstirred solution of(4S,5R)-4-hydroxy-5-(hydroxymethyl)dihydrofuran-2(3H)-one (3.30 g, 25.0mmol) and imidazole (10.2 g, 150 mmol) in DMF (35 mL). The mixture wasstirred for 1 h at 0° C. then at rt for 40 h. The reaction was quenchedwith water and the mixture extracted three times with EtOAc. The organicphase was dried (Na₂SO₄), filtered and concentrated, and the product wasisolated by silica gel column chromatography eluted with a gradient ofisohexane and 0 to 10% EtOAc. Mixed fractions were purified again bysilica gel column chromatography eluted with toluene, which gave thetitle compound (11.1 g, 94%).

Step b)(3S,4R,5R)-3-fluoro-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)-dihydrofuran-2(3H)-one(1b)

A 1M solution of lithium bis(trimethylsilyl) amide (2.18 g, 13.0 mmol)was added dropwise during 10 min to a solution at −70° C. of 1a (4.45 g,10.0 mmol) and NFSI (4.73 g, 15.0 mmol) in dry THF (50 mL). The mixturewas stirred for 90 min at −70° C., then added to a saturated solution ofammonium chloride and cracked ice. The mixture was extracted three timeswith EtOAc, the organic phase was dried (Na₂SO₄), filtered andconcentrated, and the product was isolated by silica gel chromatographyeluted with a gradient of isohexane and 0 to 5% EtOAc. Yield 4.63 g,67%.

Step c)(3S,4R,5R)-3-chloro-3-fluoro-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)-dihydrofuran-2(3H)-one(1c)

A 1M solution of lithium bis(trimethylsilyl) amide was added drop wiseduring 10 min to a solution at −70° C. of 1b (3.08 g, 6.65 mmol) andN-chlorosuccinimide (1.07 g, 7.99 mmol) in dry THF (25 mL). The mixturewas stirred for 90 min at −70° C., then added to a saturated solution ofammonium chloride and cracked ice. The mixture was extracted three timeswith EtOAc, the organic phase was dried (Na₂SO₄), filtered andconcentrated, and the product was isolated by silica gel chromatographyeluted with a gradient of isohexane and 0 to 5% EtOAc. Yield 2.40 g,73%.

Step d)(3S,4R,5R)-3-chloro-3-fluoro-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)-tetrahydrofuran-2-ol(1d)

A 1M solution of DIBAL (2.23 g, 15.7 mmol) in DCM was added dropwiseunder argon to solution at −70° C. of 1c (5.20 g, 10.5 mmol) in drytoluene (50 mL). The mixture was stirred for 2 h at −70° C., then thetemperature was raised to −30° C. and the reaction was quenched with 2mL MeOH and then added to a mixture of Rochelle salt and crashed ice.The mixture was stirred for 30 minutes and then extracted three timeswith EtOAc. The organic phase was dried (Na₂SO₄), filtered andconcentrated under reduced pressure. The product was isolated by silicagel column chromatography eluted with a gradient of isohexane and 0 to10% EtOAc. Yield 5.22 g, 85,%.

Step e)(2S,3S,4R,5R)-3-chloro-3-fluoro-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-2-ylmethanesulfonate (1e)

Mesyl chloride (688 mg, 6.00 mmol) was slowly added to a cooled solutionof 1d (2.00 g, 4.01 mmol) and TEA (608 mg, 6.00 mmol) in DCM (20 mL).The mixture was stirred for three hours at RT, then diluted with EtOAc(80 mL), washed with saturated NaHCO₃ (aq), HCl, water and with brine.The organic phase was dried (Na₂SO₄), filtered and concentrated. Thecrude product was dried in vacuo and then was used in the next stepwithout further purification.

Step f)1-((2R,3S,4R,5R)-3-chloro-3-fluoro-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione(1f)

A suspension of uracil (699 mg, 6.24 mmol) and ammonium sulfate (25.8mg, 0.195 mmol) in hexamethyldisilazane (HDMS) (40 mL) was refluxedovernight. The solvent was removed in vacuo and the residue wasdissolved in DCM (60 mL). The 1e (2.25 g, 3.90 mmol) was added underargon and then the TMS triflate was added slowly. The mixture wasstirred for 10 minutes at RT and then refluxed for 4 hours. The mixturewas added to cooled sodium hydrogen carbonate solution and extractedthree times with EtOAc. The organic phase was washed with brine anddried over sodium sulfate. The solution was evaporated under reducedpressure and the mixture was purified by silica gel chromatography withisohexane and 20 to 50% ethyl acetate, which gave the two compoundsdiTIPS (1.29 g, 56%) and monoTIPS (390 mg, 23%).

Step g)1-((2R,3S,4R,5R)-3-chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione(1g)

A solution of 1f (1.27 g, 2.14 mmol) in 80% acetic acid was stirred at80° C. for 18 h, then concentrated and co-evaporated with toluene. Theresidue was dissolved in dry THF (10 mL), triethylamine trihydrofluoridewas added (1.38 g, 8.56 mmol) and the mixture was evaporated onto silicaand purified by silica gel column chromatography eluted with DCMincluding 0 to 10% MeOH. The mixed fractions were purified by HPLC on aHypercarb column eluted with 10 to 20% acetonitrile and 10 mmol ammoniumacetate, which gave the title compound (19 mg, 3.2%). MS 281.2 [M+H]⁺.

¹H NMR (500 MHz, DMSO) δ 10.39 (s, 1H), 7.87 (d, J=8.1 Hz, 1H), 6.74 (s,1H), 6.22 (d, J=16.1 Hz, 1H, 7), 5.73 (d, J=8.1 Hz, 1H), 5.52 (s, 1H),4.21 (dd, J=19.6, 9.2 Hz, 1H), 3.87-3.77 (m, 2H), 3.64 (dd, J=12.7, 2.8Hz, 1H).

¹³C NMR (126 MHz, DMSO) δ 162.76, 150.26, 139.06, 115.71, 113.71,102.28, 86.98, 86.69, 81.01, 73.28, 73.14, 58.19.

EXAMPLE 2

(2S)-isopropyl2-(((((2R,3R,4S,5R)-4-chloro-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(2)

A 1M solution of tert-butyl magnesium chloride (0.22 mL, 0.22 mmol) wasslowly added under argon to a solution of sugar 1g (28 mg, 0.1 mmol) inTHF (1.5 mL). The suspension was stirred for one h at 0° C., then DMPU(0.5 mL) was added followed by addition of a solution of (2S)-isopropyl2-(((perfluorophenoxy)phenoxy)phosphoryl)amino)propanoate (57 mg, 0.12mmol) (prepared as described in WO2011/123672) in THF (0.5 mL) at 0° C.during ˜5 min. The mixture was stirred for 5 h at 0° C., then allowed toattain RT and was quenched with saturated ammonium chloride solution.The mixture was extracted three times with EtOAc. The organic phase wasdried (Na₂SO₄), concentrated under reduced pressure and the product wasisolated by HPLC. (Gemini NX 30 mm 20 to 60% acetonitrile 10 mmolammonium acetate Gradient 17 minutes and flow 40 ml per minute. Thetitle compound was isolated as a single isomer at the phosphorus atom.Yield 22 mg, 40%. MS ES+ 550.4 [M+H]⁺, ES− 547.9 [M−H]⁻.

¹H NMR (500 MHz, DMSO) δ 1.15 (d, 6H), 1.23 (d, 3H), 3.80 (tq, 1H), 4.04(m, 1H), 4.31 (m, 3H), 4.86 (hept, 1H), 5.63 (dd, 1H), 6.09 (dd, 1H),6.24 (d, 1H), 6.66 (d, 1H), 7.21 (m, 3H), 7.38 (m, 2H), 7.58 (d, 1H),11.63 (m, 1H).

¹³C NMR (126 MHz, DMSO) δ 19.64 (d), 21.26, 21.30, 49.67, 64.32, 67.89,74.42 (d), 78.81, 87.60 (m), 102.27, 113.96 (d), 119.96 (d), 124.52,129.56, 139.91, 150.01, 150.53 (d), 162.52, 172.45 (d).

³¹P NMR (162 MHz. DMSO) δ 3.76.

¹⁹F NMR (376 MHz. DMSO) δ −119.05.

EXAMPLE 3

Step a)(2R,3R,4S,5R)-4-chloro-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ylacetate (3a)

4-methoxytrityl chloride (133 mg, 0.43 mmol) was added to a solution ofcompound 1f (81 mg, 0.29 mmol) in pyridine (25 mL). The resultingmixture was stirred at room temperature for 40 h, the diluted with DCMand washed with NaHCO₃. The organic phase was concentrated and theresidue purified by column chromatography on silica gel, which gave thetitle compound (144 mg, 90%).

The afforded compound was dissolved in dry pyridine (1.4 mL), Ac₂O (29μL, 0.31 mmol) was added and the solution was stirred at rt. After 2 h,MeOH was added, the mixture was concentrated and extracted with DCM (×3)and the combined organic layers were washed with sat. aq. NaHCO₃,Na₂SO₄, concentrated and co-evaporated once with THF.

The residue was taken up in 80% HOAc (35 mL) and stirred at 45° C. for 3h, then concentrated. The residue was purified by column chromatographyon silica gel, which gave the title compound (69 mg, 33%).

Step b) Lithium((2R,3R,4S,5R)-4-chloro-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyltriphosphate (3b)

A freshly prepared solution of 2-chloro-1,3,2-benzodioxaphosphin-4-one(64 mg, 0.31 mmol) in anhydrous THF (280 μL) was added under nitrogen toa stirred solution of compound 3a (78 mg, 0.24 mmol) in a mixture ofanhydrous pyridine (560 μL) and anhydrous THF (560 μL). The mixture wasstirred at room temperature under nitrogen for 15 minutes, then apreviously prepared solution of tributylammonium P₂O₇ (146 mg, 0.27mmol) and tributylamine (127 μL, 0.53 mmol) in anhydrous DMF (560 μL)was added under nitrogen. The afforded solution was stirred foradditional 15 minutes at room temperature under nitrogen, then I₂ (123mg, 0.48 mmol) was added as a solution in pyridine/water (98/2, v/v)(1.1 mL) and the reaction mixture was stirred for 15 minutes. Excessiodine was destroyed by addition of ˜19 drops of a 5% aqueous solutionof Na₂SO₃ and the reaction solution was concentrated. The residue wastaken in water/acetonitrile (95:5) (5 mL) and left shaking at roomtemperature for 30 minutes. Concentrated ammonia (10 mL) was added andthe reaction mixture stirred for 1½ h at room temperature, thenconcentrated and the residue dissolved in water/acetonitrile (95:5, 5mL) and freeze dried.

The crude material ˜430 mg, was dissolved in 10% MeCN/water (3 mL) andfiltered and purified by HPLC on a Gilson instrument using a PhenomenexLuna 5μ NH₂ (150×21.2 mm) column.

Solvent A: 95% water:5%/acetonitrile: 0.05M ammonium bicarbonate

Solvent B: 95% water:5% acetonitrile: 0.8M ammonium bicarbonate

Gradient: 0% B to 50% B in 30 min.

The NTP fractions were pooled and concentrated, the residue wasdissolved in 10% MeCN/water and freeze dried. The afforded solids weretaken up in 10% MeCN/water, insolubles were filtered off through 0.45 μmfrit filters and the clear filtrate was evaporated to dryness, dissolvedin water/acetonitrile (95:5), passed through Dowex-Li⁺ and freeze-driedwhich gave the title compound (39.3 mg, 28%).

¹H NMR (500 MHz, D₂O) δ 7.87 (d, J=8.2 Hz, 1H), 6.41 (d, J=15.9 Hz, 1H,1), 5.98 (d, J=8.2 Hz, 1H), 4.56 (dd, J=19.1, 9.4 Hz, 1H, 5), 4.35(dddd, J=42.1, 12.3, 5.1, 2.2 Hz, 3H), 4.19 (d, J=9.4 Hz, 1H, 8).

¹³C NMR (126 MHz, D₂O) δ 165.94, 151.67, 140.78, 114.54, 112.55, 103.12,87.95, 87.62, 79.45, 79.38, 73.16, 73.02, 62.60, 62.56.

EXAMPLE 4

(2S)-cyclohexyl2-(((((2R,3R,4S,5R)-4-chloro-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(4)

t.BuMgCl (13.7 mg, 0.12 mmol) was added under N₂ at 0° C. to a solutionof nucleoside 1g (15 mg, 0.053 mmol) in dry THF (2 mL). The resultingsuspension was stirred for 1 h at 0° C. then DMPU (0.5 ml) was addedfollowed by dropwise addition of a solution of (2S)-cyclohexyl2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (33 mg, 0.067mmol) in THF (0.5 mL) keeping the temperature at 0° C. After 4 h, NH₄Cl(sat. aq.) was added and the mixture was extracted three times withEtOAc. The combined organic extracts were washed with water and brine,then dried (Na₂SO₄) and concentrated under reduced pressure. Theafforded residue was purified using Biotage (SNAP 25 g) eluted with agradient of DCM/MeOH followed by further purification using WatersGemini nx C18 colon, pH 7. Appropriate fractions were pooled,concentrated and co-evaporated from water, then freeze dried from MeCNand water, which gave the title compound as a single stereoisomer at thephosphorus atom as a white powder, (9.9 mg, 31.4%). LC-MS 590.09 [M+H]⁺.

The following compounds were synthesised as single stereoisomers at thephosphorus atom by phosphorylation of nucleoside 1g with the indicatedphosphorylating agent using the procedure of Example 4:

Ex Structure P-agent Yield MS (ES) 5

I-32 4.6 mg, 15% 564.1 [M + H]⁺ 6

I-33 11 mg, 38% 536.08 [M + H]⁺ 7

I-34 5.3 mg, 23%. 589.93 [M − H]⁻ 8

I-35 12 mg, 39% 564.32 [M + H]⁺ 9

I-37 4.5 mg, 19% 594.0 [M + H]⁺ 10

I-38 9.0 mg, 34% 580.0 [M + H]⁺ 11

I-39 12 mg, 43% 580.1 [M + H]⁺ 12

I-40 5 mg, 7.7% 590.0 [M + H]⁺ 13

I-41 6.5 mg, 14% 564.0 [M + H]⁺ 14

I-42 2.1 mg, 4.5% 549.9 [M + H]⁺ 15

I-43 17 mg, 32% 604.0 [M + H]⁺ 16

I-44 16 mg, 29% 604.0 [M + H]⁺ 17

I-45 9.7 mg, 18% 604.0 [M + H]⁺

EXAMPLE 18

Step a)1-((2R,3S,4R,5R)-3-Chloro-3-fluoro-4-hydroxy-5-((((6-nitro-2-oxido-4H-benzo[d][1,3,2]dioxaphosphinin-2-yl)oxy)methyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione(18a)

Nucleoside 3a (69 mg, 0.21 mmol) was dissolved in a mixture ofacetonitrile/dichloromethane: 2.7/1.3 (˜4 mL) and the solution wascooled to −20° C. under nitrogen. To the solution was added Et₃N (77 μL,0.56 mmol) followed by2-chloro-6-nitro-4H-benzo[d][1,3,2]dioxaphosphinine (125 mg, 0.54 mmol)prepared as a solution in DCM (1.34 mL; 2 mmol was diluted to 5 mL toget stock solution). The cooling bath was removed and the reactionstirred at room temperature. After 1½ h the reaction was cooled to −5°C. and a solution of Oxone®, (0.855 mmol) in water (4.0 mL) was addedand the two-phase system was vigorously stirred for 15 min. The mixturewas then extracted with EtOAc, the phases were separated and the organicphase was washed with cold water (2×), dried (Na₂SO₄). concentrated andco-evaporated from heptane/DCM, LCMS 536 [M+H]. This crude material wastaken to next step.

Step b)((2R,3R,4S,5R)-4-chloro-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyltrihydrogen diphosphate (18b)

Compound 18a was co-evaporated once with dry DMF, then dissolved in dryDMF (2.2 mL) and bis-tributylamine phosphate (0.25 mmol, 0.5 mL, 0.5M inDMF) was added under nitrogen. The solution was stirred ˜17 h at roomtemperature, then concentrated in vacuum and a few mL of water was addedfollowed by addition of conc. ammonia (25-30 mL) and a THF (1-2 mL) andthis mixture was stirred at room temperature. After 2 h. most of the NH₃was removed by evaporation and the residue was extracted with DCM (4×40mL). The water layer was concentrated and the residue dissolved in 10%MeCN/Milli Q water. Insolubles were filtered off and the filtrate wasconcentrated to dryness.

The afforded residue was dissolved in 10% MeCN/water (1.5 mL), loadedonto an active carbon column (0.85×3.00 cm) and eluted with 10%MeCN/Milli Q water. Appropriate fractions were pooled, concentrated,co-evaporated with MeCN (×2) and finally dried on a freeze dryer. Thecrude residue (76 mg) was dissolved in 10% MeCN/Milli Q water (1 mL) andpurified by semi-preparative HPLC on a Luna NH₂ column on Gilson machineusing a gradient (30 mL/min) from 0% B to 30% B over 20 min (Solvent A:0.05M ammonium bicarbonate, 5% acetonitrile, Solvent B: 0.8M ammoniumbicarbonate, 5% acetonitrile). Appropriate fractions were pooled andconcentrated to dryness, the residue dissolved in Milli Q water withsome MeCN and freeze dried. The fluffy residues were taken up in 10%MeCN in Milli Q water, the suspension was filtered through a 0.2 μmfilter and the clear filtrates were pooled and freeze dried, which gavethe title compound (28.6 mg, 36%). LCMS ES⁻ 438.8 [M−H]⁻.

EXAMPLE 19 Alternative Route to Compound 1g

Step a)(3S,4R,5R)-3-Chloro-3-fluoro-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)-tetrahydrofuran-2-ylacetate (19a)

A 1M solution of Li(O-t-Bu)₃AlH in THF (39 mL, 39 mmol) was addeddropwise under argon at −35° C. to solution of compound 1c (16.3 g, 32.8mmol) in THF (120 mL). The mixture was stirred for 1 h at −35° C., thenat rt for 1 h. The mixture was cooled to −25° C., DMAP (4.00 g, 32.8mmol) was added and the mixture was stirred for 15 minutes, then aceticanhydride (33.5 g, 328 mmol) was added drop wise and the mixture wasstirred 2 h. The mixture was allowed to come to 0° C. and EtOAc (200 mL)and water (200 mL) were added. The phases were separated and the waterphase was extracted with EtOAc (×2). The combined organic phases werewashed with water (×2) and with brine (×1). The organic phase was dried(Na₂SO₄), filtered and concentrated under reduced pressure. The residuewas co-evaporated twice with toluene and the product was purified bychromatography on silica gel with eluted with isohexane and 2 to 6%EtOAc, which gave the title compound (17.1 g, 96%).

Step b)(3S,4R,5R)-3-Chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-ylacetate (19b)

Triethylamine tri hydrofluoride (20.5 g, 126 mmol) was added to astirred solution of compound 19a (17.0 g, 31.4 mmol) in acetonitrile(115 mL) and THF (23 mL). The mixture was stirred for 72 h at rt, 20 hat 50° C. and then at rt overnight. The solution was concentrated onsilica (60 g) and purified by silica gel chromatography eluted with agradient of isohexane and EtOAc, which gave the title compound (68.0 g,85%).

Step c)(2R,3R,4S)-5-Acetoxy-2-((benzoyloxy)methyl)-4-chloro-4-fluorotetrahydrofuran-3-ylbenzoate (19c)

Triethylamine (10.8 g, 107 mmol) was added to a stirred solution ofcompound 19b (6.80 g, 26.8 mmol) under ice cooling followed by drop wiseaddition of benzoyl chloride (9.41 g, 66.9 mmol).

The mixture was allowed to attain rt and stirred overnight. EtOH (5 mL)was added and the mixture was stirred for 30 minutes, then concentratedin vacuo. Water was added and the mixture was extracted with EtOAc (×3).The organic phase was washed with water and brine, dried (Na₂SO₄),filtered and concentrated under reduced pressure. The product waspurified by silica gel chromatography eluted with a gradient ofisohexane and EtOAc, which gave the title compound (10.1 g, 86%).

Step d)((2R,3R,4S)-3-(Benzoyloxy)-4-chloro-4-fluoro-5-hydroxytetrahydrofuran-2-yl)methylbenzoate (19d)

Ethanolamine (1.55 g, 25.4 mmol) was added to a stirred solution ofcompound 19c (10.1 g, 23.0 mmol) in EtOAc (100 mL) and DMSO (50 mL). Themixture was stirred at rt for 72 h, then diluted with diethyl ether (300mL) and EtOAc (300 mL) and washed with water (×4). The combined waterphases were extracted with EtOAc then the EtOAc phase was washed withbrine (×2). The combined organic phases were dried (Na₂SO₄), filteredand concentrated under reduced pressure. The product was purified bysilica gel chromatography eluted with a gradient of DCM with and EtOAc,which gave the title compound (7.50 g, 82%).

Step e)((2R,3R,4S)-3-(benzoyloxy)-4-chloro-4-fluoro-5-((methylsulfonyl)oxy)tetrahydrofuran-2-yl)methylbenzoate (19e)

Et₃N (3.54 mL, 25.4 mmol) was added at −15° C. under N₂ to a solution ofcompound 19d (8.36 g, 21.2 mmol) in dry DCM (100 mL) followed byaddition of MsCl (1.97 mL, 25.4 mmol). The reaction mixture was stirredat −15° C. for 2 h, then poured into HCl (80 mL, 1M, aq.). The phaseswere separated and the aqueous layer was extracted with DCM. Thecombined organic extracts were washed with NH₄Cl (sat. aq.) dried(MgSO₄) and concentrated under reduced pressure to give the titlecompound (9.86 g, 98%) as a clear oil.

Step f)((2R,3R,4S,5R)-3-(benzoyloxy)-4-chloro-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluorotetrahydrofuran-2-yl)methylbenzoate (19f)

Uracil (3.09 g, 27.5 mmol) and ammonium sulfate (48.5 mg, 0.367 mmol)was heated to reflux under N₂ in HMDS (49.3 mL, 236 mmol) for 16 h. Thereaction mixture was cooled to rt, concentrated under reduced pressureand dried in vacuo. The residue in dry DCE (50 mL) was added under N₂ toa solution of compound 19e(8.68 g, 18.4 mmol) in dry DCE (75 mL). TMSOTf(6.12 g, 27.5 mmol) was slowly added under N₂ to the solution. After theaddition, the reaction mixture was heated to 80° C. for 5 h and then at65° C. for 16 h.

The reaction mixture was cooled to rt, quenched with NaHCO₃ (sat. aq.),filtered and extracted twice with DCM. The combined organic extractswere dried (MgSO₄) and concentrated under reduced pressure. EtOAc andDCM was added and the formed precipitate was collected by filtrationwhich gave the pure β-isomer (660 mg, 7.4%). The filtrate was evaporatedonto silica and purified by flash chromatography (hex:EtOAc 2:1-1:1),which gave the title compound as a mixture with the α-isomer, α:β>5:95(942 mg, 11%).

Step e)1-((2R,3S,4R,5R)-3-chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione(19g)

Compound 19f (670 mg, 1.37 mmol) was suspended in NH₃ (7N in MeOH).After 30 min, EtOH (5 mL) was added and the suspension was stirred atit. After an additional hour, the suspension went into solution and thenreaction mixture was stirred at rt for 15 h. The solvents wereevaporated under reduced pressure and the afforded residue was purifiedby flash chromatography (DCM:MeOH 10:1) which gave the title compound(380 mg, 99%) as a white solid. LC-MS ES− 279.31 [M−H]⁻.

The following compounds were synthesised as single stereoisomers at thephosphorus atom by phosphorylation of nucleoside 1g with the indicatedphosphorylating agent using the procedure of Example 4:

Ex Structure P-agent Yield MS (ES) 20  

I-46 67 mg, 32% 580.26 [M + H]⁺ 21¹

I-47 12 mg, 14% 609.9 [M + H]⁺ 22  

I-48 54 mg, 24% 578.0 [M + H]⁺ 23²

I-49 7.1 mg, 12% 549.94 [M + H]⁺ 24  

I-50 31 mg, 28% 601.2 [M + H]⁺ 25  

I-51 37 mg, 18% 580.1 [M + H]⁺ ¹No DMPU present in reaction mixture ²Anadditional 0.8 Eq of the phosphorylating agent (I-49) was added after 18h

EXAMPLE 26

Step a)(2R,3R,4S,5R)-4-Chloro-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl4-methylbenzoate (26a)

Nucleoside 1g (253 mg, 0.9 mmol) was dissolved in pyridine (5 ml) andDCM (5 ml). Triethylamine (630 μl, 4.52 mmol) was added and the mixturewas cooled on an ice bath. After 15 min, 4-methylbenzoyl chloride (300μl, 2.27 mmol) was added and the mixture was stirred with cooling for 10min then at 22° C. for 90 min. NaHCO₃ (aq) was added and the mixture wasdiluted with DCM and washed with 1M HCl (aq)×3, dried (Na₂SO₄) andconcentrated. The residue was purified by column chromatography onsilica eluted with petroleum ether/EtOAc (3:1) which gave the titlecompound (279.2 mg, 60%).

Step b)4-Amino-1-((2R,3S,4R,5R)-3-chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)pyrimidin-2(1H)-one(26b)

Compound 26a (279 mg, 0.54 mmol) was dissolved in pyridine (5 mL),molecular sieves (4 Å, half spoon) was added and the mixture was stirredon an ice bath for 15 min. Phosphorus oxychloride (200 μl, 2.18 mmol)was added and after 5 min 1,2,4-1H-triazole (373 mg, 5.4 mmol) wasadded. The mixture was stirred with cooling for 15 min then at 22° C.for 5 h. Ammonia (32%, 10 mL, 82.2 mmol) was added and the mixture wasstirred overnight at 22° C. The mixture was concentrated, dissolved inwater and washed with EtOAc×2. The combined organic layers wereextracted with water, the combined water extracts were concentrated, andthe residue was purified by column chromatography on silica eluted withDCM/MeOH (8:2) which gave the title compound (139 mg, 83%). MS ES+ 279.9[M+H]⁺.

Step c) (2S)-Isopropyl2-(((((2R,3R,4S,5R)-5-(4-amino-2-oxopyrimidin-(2H)-yl)-4-chloro-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(26c)

Compound 26b (27.4 mg, 0.1 mmol) was dissolved in dry THF (6 mL)containing molecular sieves, and the mixture was stirred at 22° C. for30 min then 2M tert-butylmagnesium chloride in THF (0.11 ml) was addedand the mixture was stirred another 30 min. (2S)-Isopropyl2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (51.4 mg,0.11 mmol) was added and the mixture was stirred for 15 h, then dilutedwith EtOAc, washed with NaHCO₃ (aq), dried (Na₂SO₄) filtered andconcentrated. The residue was purified by YMC-silica eluted with agradient of DCM:MeOH (95:5→90:10). Appropriate fractions were pooled andconcentrated.

The residue was purified by preparative HPLC using a Gemini C18 columneluted with a gradient of acetonitrile/water (pH 7, 0.01M NH₄OAc,20-40%). The product was concentrated then purified on a fluorophenylcolumn eluted with a gradient of MeOH/water (pH 7, 0.01 M NH₄OAc,33-50%). The product was collected, dissolved in acetonitrile/water(1:4) and lyophilized which gave the title compound as a singlestereoisomer at the phosphorus atom (13 mg, 24%) LC-MS 548.9 [M+H]⁺.

EXAMPLE 27

Step a)N—(—((2R,3S,4R,5R)-3-chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)isobutyramide(27a)

Isobutyric anhydride (118 mg, 0.746 mmol) was added at 58° C. to asolution of nucleoside 26b (139 mg, 0.497 mmol) in dioxane (1.7 mL) andwater (0.19 mL). The solution was stirred for 3 h at 58° C., thenconcentrated. The residue was dissolved in 20% EtOH in DCM and washed(×4) with sat.aq. NaHCO₃/brine 30:70 v/v, dried (Na₂SO₄) filtered andconcentrated. The residue was purified by column chromatography onsilica gel eluted with a gradient of EtOH/DCM (2→8%), which gave thetitle compound as a solid (62 mg).

Step b)(2R,3R,4S,5R)-4-chloro-4-fluoro-5-(4-isobutyramido-2-oxopyrimidin-1(2H)-yl)-2-(((4-methoxyphenyl)diphenylmethoxy)methyl)tetrahydrofuran-3-ylisobutyrate (27b)

4-Methoxytrityl chloride (65.7 mg, 0.177 mmol) was added to a solutionof compound 27a (62 mg, 0.177 mmol) in pyridine (1.1 mL) and theresulting mixture was shaken at room temperature for about 6 h, thenadditional 4-methoxytrityl chloride (16 mg, 0.3 eq.) was added and themixture was shaken for further 18 h. Isobutyric anhydride (33.6 mg,0.212 mmol) was added and the solution was shaken at rt for 4 h. Thereaction was quenched with MeOH, then concentrated and extracted withDCM (×3)/sat. aq. NaHCO₃. The organic phase was dried (Na₂SO₄) filteredand concentrated and the residue was co-evaporated twice with tolueneand twice with THF. The afforded solid residue was taken directly to thenext step.

Step c)(2R,3R,4S,5R)-4-Chloro-4-fluoro-2-(hydroxymethyl)-5-(4-isobutyramido-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-3-ylisobutyrate (27c)

Compound 27b (123 mg, 0.177 mmol) was dissolved in 80% AcOH (25 mL) andTHF (5 mL) and the solution was stirred at 45° C. for 2 h, thenconcentrated and co-evaporated with THF (×3) and toluene (×1). Theresidue was purified by column chromatography on silica geleluted with agradient of 0→4% EtOH in DCM, which gave the title compound (36 mg,48.5% over 3 steps). LC-MS 420.0 [M+H]⁺.

Step d)(((2R,3R,4S,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4-chloro-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)triphosphoricacid (27d)

Compound 27c (36.0 mg, 0.086 mmol) was dissolved in a mixture ofMeCN/DCM: 1.06/0.54 (˜1.6 mL) and the solution cooled to −20° C. undernitrogen. Et₃N (31.1 μL, 0.223 mmol) was added to the solution followedby addition of a solution of2-chloro-6-nitro-4H-benzo[d][1,3,2]dioxaphosphinine (50.1 mg, 0.214mmol) in DCM (0.71 mL). The cooling bath was removed and the reactionwas stirred at room temperature for 1½ h. The reaction was cooled to −5°C. and a solution of Oxone® (0.343 mmol in water (1.73 mL) was added andthe two-phase system vigorously stirred for 15 min. The mixture wasextracted with ethyl acetate, the organic phase was washed with coldwater (2×), dried (Na₂SO₄) and concentrated. The residue wasco-evaporated once with toluene and once with dry DMF, then dissolved indry DMF (1 mL). Tributylamine pyrophosphate (0.1 mmol, 54.6 mg) wasadded under nitrogen and the solution shaken for ˜18 h at roomtemperature, then concentrated. 30% MeCN/H₂O (˜20 mL) was added to theresidue and the solution was shaken for 20-25 min at rt. The volatileswere evaporated and the residual oil-solid mix was dissolved in conc.ammonia (10-15 mL) and shaken at room temperature for about 5 h.

Most of the NH₃ was removed by evaporation then the residue wasextracted with DCM (4×40 mL). The organic extracts were discarded andthe water layer was concentrated. The residue was dissolved in 5% MeCNin water (1.5-2.0 mL) and loaded onto an active carbon column(0.85×2.5). The column was washed with 5% MeCN in water and 6-7 mL ofeluent was collected and concentrated and freeze dried. The residue wasdissolved in 5% MeCN/water (1.6 mL) and purified by semi-preparativeHPLC using a Phenomenex Luna 5μ NH₂ column on Gilson machine eluted witha gradient (30 mL/min) from 0% B to 40% B over 30 min (Solvent A: 0.05Mammonium bicarbonate, 5% acetonitrile; Solvent B: 0.8M ammoniumbicarbonate, 5% acetonitrile). Appropriate NTP fractions were pooled andconcentrated to dryness, the residue was dissolved in MQ water with 5%MeCN and freeze dried. The residue was taken up in 5% MeCN in MQ water(4-5 mL) and the suspension was filtered through a 0.45 μm filter andthe filtrate was concentrated. The residue was dissolved in 5% MeCN inwater (0.5 mL) and applied onto a short Li+ Dowex column (6×1 cm) andwashed with the 5% MeCN in water. The first ˜10 mL was pooled,concentrated and freeze dried, which gave the title compound (11.7 mg,30%) in 89% purity containing 6.6% NDP according to PI analysis. MS ES+519.9 [M+H]⁺.

EXAMPLE 28 Alternative Route to Compound 2

Step a) (4S,5R)-4-Hydroxy-5-(hydroxymethyl)dihydrofuran-2(3H)-one (28a)

Deoxy-D-ribose (400.0 g, 2.98 mol) was dissolved in water (1.6 kg) undernitrogen and the solution cooled to 3-7° C. Bromine (800 g, 10.0 mol,3.36 eq.) was added at 3-7° C. while stirring over a period ofapproximately 2 hours and the stirring was continued at 3-7° C. forapproximately 1 hour. The reaction mixture was gently warmed to 20-25°C. and then stirred for approximately 20 hours.

The reaction mixture was cooled to −5 to −7° C. and a solution of sodiumhydroxide (27.65%, 720 g, 1.67 eq.) was added while keeping the reactiontemperature at −3 to −7° C. The temperature was then adjusted to 0-5° C.and aqueous sodium hydroxide (9%, 470 g, 1.06 mol, 0.35 eq. was added at0-5° C. to obtain a final pH=1.40.

The water was distilled off at reduced pressure using a scrubber(cooled, 14% sodium hydroxide, 0.9 L), finally at p<5 mbar and 50° C. Inorder to remove residual water from the product, 2-propanol was addedportion wise to the residue followed by azeotropic distillation atreduced pressure. The final water content was determined by KF titrationto be less than 1%. 2-Propanol (400 mL) was added to the residue and themixture followed by filtration. The filter cake was washed with2-propanol (1 L). The solvent was distilled off at reduced pressure.Toluene (400 mL) was added and distillation was resumed in order toremove residual 2-propanol and possibly more water. A residue of 474.6 g(120% yield) was obtained.

Step b)(4S,5R)-4-((Triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)dihydrofuran-2(3H)-one(28b)

Compound 28a (470.9 g, 2.97 mol) was dissolved in DMF (1.2 L) and cooledto 10-15° C. Imidazole (707.0 g, 10.4 mol, 3.5 eq.) was added and thetemperature of the mixture was adjusted to 3-7° C. TIPS-Cl (1145 g, 5.94mol, 2.0 eq.) was added with cooling to 3-7° C. over a period of 2hours. The reaction mixture was stirred at 3-7° C. for another ½ h, thengently warmed to 20-25° C. and stirred for 20 h. The progress of thereaction was monitored as follows: A sample of the reaction mixture wasdiluted 10 times with dry DMF, N;O-bis(trimethylsilyl)trifluoroacetamide(0.25 mL) was added to 0.5 mL of the sample in DMF and analyzed by GC.If the reaction was not complete the necessary amount of TIPS-Cl wascalculated and added and the stirring continued for another 20 hours.

When the reaction was completed, methanol (50 mL) was added and themixture was stirred for ½-1 hour at 20-25° C. Water (1.2 kg) was addedand the temperature of the mixture was adjusted to 15-25° C. pH wasadjusted to pH 2.0-2.5 by careful addition of 36% hydrochloric acid (491g, 4.7 mol). Toluene (0.9 kg) was added and the phases were separated.The organic phase was washed twice with 5% aqueous sodium chloride (1kg). the aqueous phases were washed with toluene (0.9 kg). The organicphases were combined and dried with sodium sulfate (150 g) for minimum 1hour. The suspension was filtered on a column prepared from silica Gel60 (210 g) and toluene and the column was washed with toluene (1.1 kg).The combined filtrate was concentrated to dryness at reduced pressure at50° C. which gave the title compound (1338 g, 84.4% from crude 2a).Purity (GC): 93.9%.

Step c)(3S,4R,5R)-3-fluoro-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)-dihydrofuran-2(3H)-one(28c)

Compound 28b (450.0 g, 1.01 mol) and NFSI (348.0 g, 1.10 mol) weredissolved in Me-THF (2.2 L) under argon. The solution was cooled tobelow −75° C. and lithium bis(trimethylsilyl)amide (20.2% in THF, 1.190kg, 1.42 eq.) was added over a period of 3-4 hours. The progress of thereaction was monitored by GC, and when deemed completed, methylsulfide(6 g, 0.1 mol) was added to quench residual NFSI and the stirringcontinued for another 20-30 minutes.

The reaction mixture was transferred into aqueous 12.5% ammoniumchloride (1.7 kg) and the mixture was warmed to room temperature. Theaqueous layer (Aq. 1) was separated and the organic phase was washedwith purified water (1 L). The aqueous wash (Aq. 2) was separated andthe organic phase was secured. Aq. 1 was washed with heptanes (0.6 kg).The aqueous phase was separated and then discarded. Aq. 2 was added tothe organic phase and the mixture was stirred for 1 minute. The aqueousphase was separated and discarded. The two organic phases were combinedand concentrated at reduced pressure at 50° C. Heptanes (0.7 kg) wasadded to the residue and the resulting suspension was filtered. Thefilter cake was washed with heptanes (0.2 kg), the combined filtrate wasconcentrated at reduced pressure at 50° C., which gave 506 g crudeproduct. The crude product was dissolved in a mixture of heptanes andtoluene (0.5 L, 3:1) and purified by column chromatography on silica gel(silica gel 60, 2.5 kg and heptanes/toluene 3:1 v/v). The column waseluted with heptanes/toluene (3:1, 5.0 L), heptanes/toluene (2:1, 2.5L), heptanes/toluene (3:1, 2.5 L) and toluene (7.5 L). Fractions of ˜1 Lwere collected and fractions holding pure compound 2c were combined andconcentrated and fractions holding mixtures of compound 2c and di-fluorocompound were combined and re-purified.

The above procedure was repeated twice, starting with 450 g and 525 g ofcompound 2b. Total yield of the title compound was 877.1 g (59.2%)+104.1g (7.0%) from reworked material. Purity (GC): 92.4%.

Step d)(3S,4R,5R)-3-Chloro-3-fluoro-4-((triisopropylsilyl)oxy)-5-(((triisopropylsilyl)oxy)methyl)-dihydrofuran-2(3H)-one(28d)

Compound 28c (400.0 g, 0.86 mol) and NCS (138.0 g, 1.04 mol, 1.2 eq.)were stirred in THF (2.0 L) under argon at ˜20° C. The suspension wascooled to below −70° C. and then lithium bis(trimethylsilyl)amide (20.2%in THF, 1.150 kg, 1.6 eq.) was added over a period of 1-1.5 hours. Thereaction was monitored by GC and when deemed completed, the mixture wastransferred into a 12.5% aqueous solution of ammonium chloride (1.5 kg).The mixture was warmed to room temperature. The stirring was stopped andthe aqueous layer was separated, washed with heptanes (0.8 L) and thendiscarded.

The mother organic phase was concentrated to dryness at reduced pressureat 55° C. and then added to the heptane wash. The thus combined organicphases were washed with 5% aqueous sodium chloride. The phases wereseparated and the aqueous phase washed with heptanes (0.2 L), thendiscarded. The organic phase was concentrated at reduced pressure whichgave 440 g of crude product.

The procedure was repeated starting with 426.5 g of compound 2c whichgave 473 g of crude product.

The combined crude products were dissolved in a mixture of heptanes andtoluene (1.0 L, 2:1) and purified on a silica gel column prepared fromsilica gel 60 (2.25 kg) and heptanes/toluene 2:1 v/v. The column waseluted with: heptanes/toluene (2:1, 15 L). Fractions of ˜1 L werecollected and pure fractions of compound 2d were combined andconcentrated at reduced pressure which gave the title compound (667.3 g,75.1%).

Step e)(3S,4R,5R)-3-Chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)dihydrofuran-2(3H)-one(28e)

Compound 28d (613.0 g, 1.11 mol) was added to a 3 L glass reactor filledwith nitrogen and methanol (1.2 L) and. To the stirred emulsion wasadded 37% hydrochloric acid (368.0 g, 3.73 mol, 3.4 eq.) and the mixturewas heated to gentle reflux (73° C.). The mixture was kept at reflux for20 hours then cooled to 15-20° C. and extracted with heptanes (4×600mL). The residual methanolic solution was concentrated to dryness atreduced pressure using a water bath of 80-90° C., finally at p<35 mbar.Dioxane (600 mL) was added and distilled again as above, which gave thetitle compound (200.7 g, 98%).

Step f(2R,3R,4S)-4-chloro-4-fluoro-2-(((4-methylbenzoyl)oxy)methyl)-5-oxotetrahydrofuran-3-yl4-methylbenzoate (28f)

A solution of compound 28e (200.7 g, 1.11 mol) in dioxane (1.4 L) in a 3L glass reactor filled with nitrogen and equipped with mechanicalstirring, thermometer and an addition funnel was heated to 40 to 45° C.on a water bath. p-Toluoyl chloride (360.5 g, 2.33 mol, 2.1 eq.) wasadded whereafter triethylamine (258.3 g, 2.55 mol, 2.3 eq.) was addedduring 35 minutes so as to keep the reaction temperature below 70° C.The resulting suspension was then stirred at 65° C. for 2 hours, thencooled to 15° C. and filtered. The 800 mL filter cake was washed withdioxane (800 mL, 15° C.), leaving a white filter cake which wasdiscarded. The filtrate was concentrated at reduced pressure, finally at35 mbar using a water bath of 65° C. 2-Propanol (1.50 L) was added tothe residual oil (510 g) so as to keep the temperature of the solutionat 40-45° C. The solution was seeded and carefully allowed to cool toroom temperature. During the cooling process samples of 0.25 mL weretaken and mixed with 0.25 mL of water for pH measurements. Triethylamine(15 g) was added until pH 2.5-3.5 was obtained. Once room temperaturewas reached (one hour), the crystal suspension was cooled to 10±1° C.and kept at this temperature for 15 hours. The title product wasisolated by filtration, washed with 2-propanol (600 mL, 5-10° C.) andthen dried at 30-50° C. in an air vented oven. Yield: 374.2 g, 80%.Purity (HPLC): 99.4%. Melting point: 88.0-89.5° C. (1° C./min) crystalform change and then melts at 97-98° C.

Step g)(2R,3R,4S)-4-Chloro-4-fluoro-5-hydroxy-2-(((4-methylbenzoyl)oxy)methyl)-tetrahydrofuran-3-yl4-methylbenzoate (28g)

A 3 L reaction flask set up with mechanical stirrer, thermometer and anaddition funnel was filled with nitrogen. The flask was charged withethyl acetate (1000 g) and cooled to 10° C. Lithiumtri-tert-butoxyaluminium hydride (30% solution in THF, 35 g, 0.05 eq.)was added. Stirring at 10° C. was continued for 5-10 minutes and thencompound 28f (370.0 g, 0.88 mol) was added.

Further lithium tri-tert-butoxyaluminium hydride (30% solution in THF,933.8 g, 1.10 mol, 1.25 eq.) was added over a period of 70 minutes whilekeeping the reaction temperature at 10° C. The reaction was quenched bypouring the reaction mixture onto a quench mixture (1.45 kg (10%NaCl-10% NH₄Cl in 3M HCl)) keeping the temperature at 10-15° C. Theresulting suspension was warmed to 20-25° C. The aqueous was separatedand discarded and the organic phase was washed with acidic water (1.0L+10 mL of 3M HCl) followed by a wash with 25% sodium chloride (250 mL).The organic phase was concentrated to dryness, finally at p<35 mbar and45° C. The residue was re-dissolved in toluene (0.45 kg) and thesolution was again concentrated, at p<35 mbar and 45° C., which gave thetitle compound as an oil containing a little solid sodium chloride(412.6 g, 111%). Purity (HPLC) 97.5%.

Step h)(2R,3R,4S)-4,5-dichloro-4-fluoro-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl4-methylbenzoate (28h)

A 2000 mL reaction flask set up for mechanical stirring, temperaturemeasurement and condenser was filled with nitrogen and charged withtoluene (740 mL), compound 28g (411.5 g, 0.88 mol) and thionyl chloride(174.0 g, 1.46 mol, 1.66 equivalents). The reaction flask was placed ona water bath, pre-heated to 50° C. and DMF (0.50 mL) was added. The topof the condenser was connected a cooled scrubber (700 g of 27.65% sodiumhydroxide) and a steady flow of nitrogen was applied. The reactionstarted shortly after the DMF was added and it was followed by HPLC.After approximately three hours, the gas evolution has decreased and thetemperature was increased to 60-65° C. Heating at 60-65° C. wascontinued for further 4.5 hours after which time the sulfite esters hadvanished. The solvent and residual thionyl chloride was distilled off(500 mL) at reduced pressure using a water bath of 60-65° C. Toluene(650 mL) was added to the residual oil and the mixture was cooled to 5°C. Water (650 mL) was added and the pH was adjusted to 2.0-3.0 byaddition of 3M sodium hydroxide (40 mL) at a temperature below 10° C.The temperature was adjusted to 20-22° C. and the aqueous phase wasseparated. The organic phase was washed with 25% sodium chloride (250mL). The aqueous phases were back washed with toluene (250 mL). Thecombined organic phase was dried with magnesium sulfate (25 g) andfiltered. Evaporation of the solvent (finally at p<35 mbar and 60° C.)provided the title compound as a light brown oil (378.5 g, 97% yield).Chlorobenzene (200 g) was added to the residue and the mixture wasconcentrated using the above conditions. The residue was again dissolvedin chlorobenzene (200.0 g) and the mixture concentrated.

Step i)(2R,3R,4S,5R)-5-(4-Benzamido-2-oxo-3,4-dihydropyrimidin-1(2H)-yl)-4-chloro-4-fluoro-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl4-methylbenzoate (28i)

A 500 mL round bottom flask was charged with N-benzoylcytosine (36.6 g,170 mmol, 1.5 eq.), chlorobenzene (165 g, 150 mL) and ammonium sulfate(0.45 g, 3.4 mmol, 0.03 eq.), to this suspension was added HMDS (29.3 g,181.3 mmol, 1.6 eq.). The suspension was heated to reflux. When thereaction mixture became a clear solution, it was refluxed for additional1 h and then concentrated by distillation in vacuo at 60° C.(distillate: 150 mL). Chlorobenzene (125 mL) was added to the residue.

Residual toluene in Compound 28h (50 g, 113.3 mmol) was removed bydistillation in vacuo from chlorobenzene. The residue from thisco-evaporation was dissolved in 1,2-dichloroethane (200 mL), and thissolution was charged to the solution of silylated nucleoside inchlorobenzene. Tin(IV)chloride (59.0 g, 226.6 mmol, 2 eq.) was added andthe mixture was heated to reflux under nitrogen. The reaction mixturewas stirred at reflux for 65 h. The reaction mixture was cooled to 5°C., and ethyl acetate (99.8 g, 10 eq.) was added while keeping thetemperature at 10-12° C. Total weight of mixture: 601.7 g. A quarter ofthis mixture (150.4 g, in theory 28.3 mmol) was charged to a 250 mL 3necked round bottom flask, cooled to 5° C., and dichloromethane (147.5g, 4× vol. of EtOAc) was added together with Celite (6.25 g). A warm(approx. 60° C.) 50% NaOH solution (17.6 g, 7.76 eq.) was added to themixture in such a rate that the temperature was kept at 5-12° C. Themixture was stirred for 20 min at 10° C., then the temperature wasadjusted to 25° C. and the mixture was stirred at this temperature for30 min. The suspension was filtered on a pad of Celite (12.5 g) and thefilter cake was washed with dichloromethane (190 mL). The combinedfiltrate and washings were concentrated to dryness by distillation invacuo at 60° C. Dichloromethane (86 mL) was added to the residue thentoluene (62 mL). The content of dichloromethane was removed bydistillation in vacuo at 50° C. The resulting suspension was stirred atroom temperature for 17 h whereafter the crude title compound wasisolated by filtration. The filter cake was washed with toluene (25 mL)and the wet product was dried in an air ventilated dryer at 40° C.,which gave title compound as a solid (5.56 g, 31.7%).

Step j) (2R,3R,4S,5R)-4-Chloro-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl4-methylbenzoate (28j)

Compound 28i (15.2 g 24.5 mmol) was suspended in 65% AcOH/water (152 mL,v/v), and the suspension was heated to reflux for 20 h. The reactionmixture was allowed to cool to room temperature, then water (53 mL) wasadded and the mixture was stirred at room temperature for 1.5 h. Thesuspension was filtrated and the filter cake washed with water (2×25mL). The wet filter cake was dried in an air ventilated dryer at 40° C.for 20 h, which gave the title compound as a solid (10.8 g, 85%).

Step k)1-((2R,3S,4R,5R)-3-Chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione(28k)

Compound 28j (8.0 g, 15.5 mmol) was suspended in MeOH (80 mL),n-propylamine (9.1 g, 154.8 mmol, 10 eq.) was added and the mixture washeated to 30° C. and stirred at this temperature for 24 h. The solventswere removed by distillation in vacuo at 40° C. The residue was taken upin water (20 mL), the aqueous phase was washed with DCM (3×40 mL) andthe combined organic phases were washed with water (5 mL). The twoaqueous phases were combined, and pH adjusted to 1.0 with 3 M HCl(approx. 7 mL). The acidic aqueous phase was extracted with Me-THF (4×40mL), and the combined organic phases were concentrated to dryness bydistillation in vacuo at 40° C. Isopropyl acetate (80 mL) was added tothe residue, and the turbid mixture was concentrated in vacuo at 60° C.Isopropyl acetate (40 mL) was added and the distillation in vacuo wascontinued. Isopropyl acetate (10 mL) was added to the resulting thicksuspension. The suspension was cooled to room temperature and stirredfor 30 min. Crude title compound was collected by filtration, and thefilter cake was washed with isopropyl acetate (2×4 mL). The affordedcrude was dissolved in Me-THF (35 mL), isopropyl acetate (70 mL) wasadded and the mixture was concentrated by distillation in vacuo at 60°C. (distillate: 70 mL). Additional isopropyl acetate (30 mL) was added,and the distillation was continued (distillate: 30 mL). The suspensionwas cooled to room temperature, stirred at for 45 min and then filtered.The filter cake was washed with isopropyl acetate (2×4 mL) then dried invacuo at room temperature. The title compound was isolated in 70% yield(3.0 g). Purity (HPLC) 98.5%.

Step l) (S)-Isopropyl2-(((S)-(((2R,3R,4S,5R)-4-chloro-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy(phenoxy)phosphoryl)amino)propanoate(28)

THF (0.07% water, 12 mL) was added to Compound 28k (500 mg, 1.78 mmol)and the solution was cooled to −10° C. under nitrogen.Tert-butylmagnesium chloride, 20% wt in THF (2.20 g, 3.74 mmol, 2.1 eq.)was added by syringe over 20 min at −10° C. The syringe was rinsed with500 μL THF and the rinse was added to the reaction mixture. The formedsuspension was stirred at −10° C. for 40 min. A solution of(S)-isopropyl2-(((S)-(perfluorophenoxy)phenoxy)phosphoryl)-amino)propanoate (1.01 g,2.23 mmol, 1.25 eq.) in THF (10 mL) and DMPU (2.0 mL, 16.9 mmol, 9.5eq.) was added with a syringe at −10° C. over a period of 87 minwhereafter the reaction mixture was stirred at −10° C. for 22 h. Thereaction was quenched by addition of 1 M HCl (4.6 mL, 2.6 eq.) whilekeeping the temperature below 5° C. Toluene (20 mL) was added and themixture was heated to 25° C. and stirred at this temperature for 5 min.The phases were separated and the aqueous phase was extracted withtoluene/THF (1:1, 10 mL). The organic phases were washed with 1 M HCl(2×10 mL) and 5% Na₂CO₃ (2×10 mL). The combined basic aqueous phaseswere extracted with toluene (1×10 mL) and toluene/THF (1:1, 2×10 mL) andthe combined organic phases were washed with 25% NaCl (15 mL). Allorganic phases were then combined and the solvents removed bydistillation in vacuo at 60° C. 2-Propanol (20 mL) and n-heptane (30 mL)was added to the residue and the suspension was cooled to 5° C.overnight. The suspension was filtered and the filtrate was concentratedby distillation in vacuo at 50° C. The residue was dried on a pump for 3h which gave the title compound as a single stereoisomer at thephosphorus atom as a foam (874 mg, 89%). Purity (HPLC) of crude 91.8%.

NMR spectra obtained for compound 28 prepared in Example 28 were inagreement with spectral data of compound 2 prepared in Example 2.

NMR data for a selection of the exemplified compounds:

Compound 5

¹H NMR (500 MHz, DMSO) δ 0.81 (t, 3H), 1.12 (d, 3H), 1.24 (d, 3H), 1.49(dh, 2H), 3.83 (tq, 1H), 4.03 (m, 1H), 4.30 (m, 3H), 4.71 (h, 1H), 5.60(d, 1H), 6.11 (dd, 1H), 6.24 (d, 1H), 7.20 (dd, 4H), 7.37 (t, 2H), 7.56(d, 1H).

¹³C NMR (126 MHz, DMSO) δ 172.59, 172.55, 162.84, 162.82, 150.55,150.50, 150.21, 129.54, 124.49, 119.96, 119.92, 115.02, 113.01, 102.29,78.79, 74.45, 74.31, 72.22, 64.31, 49.72, 40.28, 40.24, 39.99, 39.90,39.82, 39.73, 39.66, 39.57, 39.49, 39.40, 39.23, 39.07, 38.90, 27.98,19.70, 19.65, 18.93, 9.30, 0.00.

Compound 9

¹H NMR (500 MHz, DMSO-d₆) δ 7.55 (d, J=7.8 Hz, 1H), 6.87 (d, J=8.4 Hz,1H), 6.84 (d, J=1.7 Hz, 1H), 6.76-6.60 (m, 2H), 6.32-6.19 (m, 1H),6.10-6.01 (m, 1H), 6.02 (s, 2H), 5.62 (d, J=8.1 Hz, 1H), 4.86 (p, J=6.3Hz, 1H), 4.37-4.15 (m, 4H), 4.07-3.97 (m, 1H), 3.79 (tq, J=10.1, 7.1 Hz,2H), 1.23 (d, J=7.1 Hz, 3H), 1.16 (d, J=6.3 Hz, 5H).

¹³C NMR (126 MHz, DMSO-d₆) δ 172.50, 147.46, 144.86, 144.81, 143.91,115.06, 115.05, 113.05, 113.05, 112.41, 112.40, 112.37, 112.37, 107.88,102.36, 102.34, 101.52, 78.74, 74.44, 74.30, 67.90, 64.28, 49.65, 40.63,40.40, 40.34, 40.27, 39.99, 39.90, 39.83, 39.73, 39.66, 39.57, 39.40,39.23, 39.07, 38.90, 21.28, 21.26, 19.72, 19.67, −0.00.

Compound 10

¹H NMR (500 MHz, DMSO-d₆) δ 7.58 (d, J=8.1 Hz, 1H), 7.28 (d, J=8.0 Hz,1H), 7.20-7.05 (m, 2H), 6.90 (td, J=7.9, 1.6 Hz, 1H), 5.60 (d, J=8.1 Hz,1H), 4.87 (dq, J=12.5, 6.2 Hz, 1H), 4.41-4.20 (m, 5H), 4.09-3.99 (m,1H), 4.00-3.77 (m, 2H), 3.79 (s, 3H), 1.79 (s, 1H), 1.22 (d, J=7.1 Hz,3H), 1.16 (d, J=6.3 Hz, 5H).

¹³C NMR (126 MHz, DMSO-d₆) δ 172.59, 172.55, 162.64, 150.28, 150.24,150.09, 139.37, 139.32, 125.29, 120.90, 120.88, 120.25, 115.03, 113.02,112.85, 102.25, 78.82, 74.38, 74.24, 67.85, 64.26, 55.59, 49.57, 40.26,40.20, 40.17, 39.99, 39.90, 39.82, 39.73, 39.66, 39.57, 39.40, 39.23,39.07, 38.90, 21.30, 21.26, 19.63, 19.58.

Compound 11

¹H NMR (500 MHz, DMSO-d₆) δ 7.57 (d, J=8.1 Hz, 1H), 7.13 (d, J=8.3 Hz,2H), 6.98-6.86 (m, 3H), 6.25 (t, J=16.6 Hz, 1H), 5.62 (d, J=8.1 Hz, 1H),4.86 (hept, J=6.2 Hz, 1H), 4.37-4.15 (m, 4H), 4.07-3.97 (m, 1H), 3.78(tq, J=10.2, 7.1 Hz, 1H), 3.72 (s, 2H), 1.23 (d, J=7.1 Hz, 3H),1.22-1.11 (m, 8H).

¹³C NMR (126 MHz, DMSO-d₆) δ 172.52, 172.48, 162.65, 155.91, 150.08,143.96, 143.91, 120.95, 120.92, 114.99, 114.42, 112.98, 102.27, 78.77,74.44, 74.30, 67.86, 64.21, 55.29, 49.65, 40.25, 40.15, 39.99, 39.90,39.83, 39.74, 39.66, 39.57, 39.40, 39.24, 39.07, 38.90, 21.30, 21.27,19.69, 19.64.

Compound 13

¹H NMR (500 MHz, DMSO-d₆) δ 0.82 (t, 3H), 1.12 (d, 3H), 1.25 (d, 3H),1.49 (m, 2H), 3.83 (dtd, 1H), 4.02 (m, 1H), 4.26 (dt, 2H), 4.34 (m, 1H),4.72 (h, 1H), 5.58 (d, 1H), 6.12 (dd, 1H), 6.26 (m, 1H), 7.20 (m, 3H),7.37 (t, 2H), 7.53 (d, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ9.35, 19.05, 19.77 (d), 28.00, 40.08, 49.72,64.32, 72.27, 74.35 (d), 78.72 (m), 102.36, 114.08 (d), 119.95 (d),124.49, 129.54, 150.53 (d), 163.41 (m), 172.62 (d).

Compound 15

¹H NMR (500 MHz, DMSO-d₆) δ 1.16 (d, 6H), 1.24 (d, 3H), 1.80 (m, 1H),1.96 (m, 1H), 2.05 (pdd, 2H), 2.26 (m, 2H), 3.49 (p, 1H), 3.81 (tq, 1H),4.04 (m, 1H), 4.30 (m, 3H), 4.86 (hept, 1H), 5.60 (d, 1H), 6.07 (dd,1H), 6.24 (d, 1H), 6.68 (d, 1H), 7.03 (m, 3H), 7.28 (t, 1H), 7.58 (d,1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 17.58, 19.65 (d), 21.26, 21.29, 29.13,49.65, 64.33, 67.87, 74.37 (d), 78.78, 102.28, 113.98 (d), 117.35 (d),117.76 (d), 122.45, 129.25, 139.49, 147.50, 150.05, 150.56, 162.56,172.48 (d).

Compound 16

¹H NMR (500 MHz, DMSO-d₆) δ 0.78 (m, 8H), 1.15 (d, 12H), 1.23 (d, 7H),1.35 (s, 6H), 3.80 (tq, 2H), 4.03 (m, 2H), 4.25 (m, 4H), 4.34 (m, 2H),4.86 (p, 2H), 5.59 (d, 2H), 6.07 (dd, 2H), 6.24 (d, 2H), 6.72 (s, 1H),7.01 (m 6H), 7.26 (t, 2H), 7.57 (d, 2H).

¹³C NMR (126 MHz, DMSO-d₆) δ 16.03, 18.82, 19.65 (d), 21.26, 21.30,24.42, 49.63, 64.29, 67.87, 74.35 (d), 78.78, 87.51, 102.30, 114.00 (d),116.92 (d), 117.60 (d), 122.06, 129.18, 139.60 (m), 148.45, 150.13,150.50 (d), 162.69, 172.47 (d).

Compound 17

¹³C NMR (126 MHz, DMSO-d₆) δ 15.52, 18.66, 19.65 (d), 21.25, 21.30,24.98, 49.67, 64.23, 67.87, 74.35 (d), 78.76, 87.49, 102.25, 113.97 (d),119.69 (d), 127.19, 139.65 (d), 142.69, 148.17 (d), 150.02, 162.52,172.46 (d).

Compound 21

¹H NMR (500 MHz. DMSO) δ 1.25 (d, 3H), 3.23 (m, 6H), 3.41 (m, 4H), 3.87(ddt, 1H), 4.04 (m, 1H), 4.31 (m, 3H), 5.02 (p, 1H), 5.61 (d, 1H), 6.20(m, 2H), 7.21 (m, 3H), 7.38 (t, 2H), 7.57 (d, 1H).

¹³C NMR (126 MHz, DMSO) δ 19.72 (d), 49.60, 58.36, 64.28, 70.33, 70.46,71.53, 74.38 (d), 78.81 (d), 102.26, 114.00 (d), 119.99 (d), 124.53,129.55, 150.03, 150.51 (d), 162.54, 172.59 (d).

Compound 24

¹H NMR (500 MHz, DMSO) δ 1.15 (dd, 6H), 1.22 (d, 3H), 3.52 (m, 1H), 3.78(tq, 1H), 4.05 (m, 1H), 4.16 (m, 1H), 4.26 (dt, 1H), 4.34 (m, 1H), 4.86(hept, 1H), 5.65 (d, 1H), 6.14 (dd, 1H), 6.23 (s, 1H), 6.27 (s, 1H),7.21 (m, 3H), 7.37 (t, 2H), 7.50 (d, 1H).

¹³C NMR (126 MHz, DMSO) δ 19.62 (d), 21.25 (d), 49.61, 63.83, 67.92,74.16 (d), 78.53, 102.42, 114.03 (d), 119.85 (d), 124.49, 129.58, 139.35(dd), 150.26, 150.56 (d), 162.87, 172.51 (d).

Compound 26b

¹H NMR (500 MHz, DMSO) δ 3.62 (d, 1H), 3.80 (m, 2H), 4.15 (dd, 1H), 5.26(s, 1H), 5.77 (d, 1H), 6.31 (d, 1H), 6.41 (s, 1H), 7.33 (s, 1H), 7.36(s, 1H), 7.73 (d, 1H).

¹³C NMR (126 MHz, DMSO) δ 58.50, 58.62, 73.62 (d), 80.48, 87.01 (m),94.50, 94.56, 114.92 (d), 140.04, 154.57, 165.42.

Compound 27d

¹H NMR (500 MHz, D₂O) δ 4.12 (d, 1H), 4.24 (ddd, 1H), 4.33 (m, 1H), 4.46(dd, 1H), 6.09 (d, 1H), 6.39 (d, 1H), 7.80 (d, 1H).

¹³C NMR (126 MHz, D₂O) δ 62.48 (d), 73.03 (d), 78.99 (d), 88.15 (d),97.04, 113.71 (d), 140.63, 157.39, 166.21.

BIOLOGICAL EXAMPLES

Replicon Assay

The compounds of formula I may be examined for activity in theinhibition of HCV RNA replication in a cellular assay aimed atidentifying compounds that inhibit a HCV functional cellular replicatingcell line, also known as HCV replicons. A suitable cellular assay isbased on a bicistronic expression construct, as described by Lohmann etal. (1999), Science vol. 285 pp. 110-113 with modifications described byKrieger et al. (2001). Journal of Virology 75: 4614-4624, in amulti-target screening strategy.

The assay utilizes the stably transfected cell line Huh-7 luc/neo(hereafter referred to as Huh-Luc). This cell line harbors an RNAencoding a bicistronic expression construct comprising the wild typeNS3-NS5B regions of HCV type 1b translated from an Internal RibosomeEntry Site (IRES) from encephalomyocarditis virus (EMCV), preceded by areporter portion (FfL-luciferase), and a selectable marker portion(neo^(R), neomycine phosphotransferase). The construct is bordered by 5′and 3′ NTRs (non-translated regions) from HCV type 1b. Continued cultureof the replicon cells in the presence of G418 (neo^(R)) is dependent onthe replication of the HCV RNA. The stably transfected replicon cellsthat express HCV RNA, which replicates autonomously and to high levels,encoding inter alia luciferase, are used for screening the antiviralcompounds.

The replicon cells are plated in 384 well plates in the presence of thetest and control compounds which are added in various concentrations.Following an incubation of three days, HCV replication is measured byassaying luciferase activity (using standard luciferase assay substratesand reagents and a Perkin Elmer ViewLux™ ultraHTS microplate imager).Replicon cells in the control cultures have high luciferase expressionin the absence of any inhibitor. The inhibitory activity of a compoundon luciferase activity is monitored on the Huh-Luc cells, enabling adose-response curve for each test compound. EC₅₀ values are thencalculated, which value represents the amount of the compound requiredto decrease the level of detected luciferase activity by 50%, or morespecifically, the ability of the genetically linked HCV replicon RNA toreplicate.

Enzyme Assay

As may be demonstrated in the replicon assay, the compounds of theinvention are metabolised by cellular kinases in target tissues to the5′-trisphosphate. It is this triphosphate which is believed to be theantivirally active species. The enzyme assay described here may be usedto confirm that compounds of the invention are antivirally active as the5′-triphosphate metabolite.

The enzyme assay measures the inhibitory effect of triphosphatecompounds in an HCV NS5B-21 (21-amino acid C-terminally truncatedversion) SPA assay (scintillation proximity assay). The assay isperformed by evaluating the amount of radiolabelled ATP incorporated byHCV NS5B-21 into newly synthesized RNA using an heterogeneousbiotinylated RNA template.

To determine IC₅₀ values the compounds are tested at variousconcentrations in a final volume of 100 μl of reaction mixture. Thereaction is stopped by addition of 0.5M EDTA solution.

The samples are transferred into flashplates precoated withstreptavidin. The incorporated radioactivity is quantified using ascintillation counter (Wallac Microbeta Trilux).

Materials & Supplier

Flashplate coated with streptavidin  PerkinElmer  Life Sciences96 well polypropylene plate  Corning Biotinylated RNA template: with a Medprobe sequence of 5′-UUU UUU UUU UAG UCA GUC GGC CCG GUU UUC CGG GCC-3′ (SEQ ID NO: 1) and biotinylated at the 5′-primer end made up to 83 μM  in 10 mM Tris-HCl, 100 mM NaCl, pH = 8.0 Enzyme: HCV NS5B-21, made up to  Replizyme 500 μg/ml in water.Nucleotides: GTP, CTP, UTP  Invitrogen Radiolabelled ³H-ATP GE Healthcare (cat. no TRK747)  0.5 M EDTA, pH = 8.0 Life  TechnologiesTris-HCl  Sigma MnCl₂  Sigma Ammonium acetate Sigma DTT (dithiothreitol)Sigma CHAPS Sigma RNase Out (cat. No 10777-019) Invitrogen DMSOCarlo Erba  Reactifs-SDSEquipment

Wallac Microbeta Trilux Perkin Elmer Life SciencesMethod

Assay Conditions

Buffer: 20 mM tris-HCl, 100 mM ammonium pH 7.5 acetate, 20 mM NaCl, 2.5mM MnCl₂, 10 mM DTT, 2 mM CHAPS, RNase Out GTP 50 μM CTP 2 μM UTP 2 μMATP 2 μM ³H-ATP (47 Ci/mmol) 0.5 μM Template: RNA-H3 83 nM Enzyme:NS5B-21 (500 μg/ml) 2 μg/ml Assay volume 100 μl

The assay should include enzyme controls (about four, containing 1 μlDMSO instead of inhibitor) and background control containing allingredients except template.

Compounds are serially diluted in DMSO on a separate dilution plate to100× the final desired assay concentrations.

Sufficient reaction mixture for the number of wells to be used is madeup according to the table below and 90 μl/well is added to a 96 wellpolyproylene plate. 1 μl of compound in DMSO from the dilution plate isadded to each well, except the enzyme control wells and backgroundcontrol wells to which 1 μl DMSO is added.

Reaction Mixture

Component μl/well 50 mM tris-HCl pH = 7.5 40 1M Ammonium acetate 10 1MMnCl₂ 0.25 0.5M DTT 2 100 mM CHAPS 2 RNase Out 0.2 1 mM GTP 5 200 μMCTP + UTP 2 NS5B-21 500 μg/ml 0.4 Template: RNA-H3, 83 μM 0.1 Templatebuffer: 10 mM tris-HCl, 100 mM NaCl pH = 8.0 28.25

Prepare an ATP cocktail containing 1.5 μl/well of ³H-ATP (45 Ci/mmol),2.0 μl well of 100 μM ATP and 6.5 μl/well of H₂O and start the reactionby adding 10 μl/well of this cocktail.

Incubate at 22° C. for 120 min.

-   Stop the reaction with the addition of 100 μl/well of 0.5M EDTA,    pH=8.0.-   Transfer 185 μl/well to the streptavidin flash plate.-   Incubate the plate over night and read the flash plate in the    Microbeta Trilux using the protocol Flash plates H3.    Treatment of results

Calculation for Inhibition:

${\%\mspace{14mu}{Inhibition}} = \frac{{CompoundCPM} - {BackgroundCPM}}{{AverageEnzymeControlCPM} - {BackgroundCPM}}$Background=Reaction buffer without template.

IC₅₀ is determined using Graphpad Prism. Plot Compound concentration inLog versus percentage inhibition. Fit the curve with nonlinearregression to the Log (Inhibitor) versus Response equation.

$Y = {{Bottom} + \frac{{Top} - {Bottom}}{1 + 10^{({X - {\log({IC}_{50})}})}}}$

Where Y is % Inhibition, X is log (inhibitor) and top and bottom are theupper and lower limits of the % Inhibition.

Biological Example 1

The inhibition of HCV replication exhibited by the compounds of theinvention were tested in the above described replicon assay. Thecompounds showed sub micromolar activity, with a cell toxicity in theHuh-Luc cell line being in excess of 50 μM. The EC₅₀ values arepresented in Table 1.

TABLE 1 Example EC₅₀ (μM) 1 >50 2 0.055 4 0.090 5 0.043 6 0.079 7 0.0558 0.044 9 0.19 10 0.045 11 0.15 12 0.18 13 0.061 14 0.076 15 0.090 160.091 17 0.11 20 0.065 21 0.21 22 0.058 23 5.1 24 1.4 25 0.1 26 0.086

Biological Example 2

The nucleotides of Examples 3 and 27 were tested in the above describedenzyme assay and the IC₅₀ values determined to be 0.72 μM and 0.089 μMrespectively.

Comparative Example 1

Sofosbuvir is marketed in several countries for the treatment of HCV,predominantly against genotypes 1 and 4. The structure of sofosbuvir is:

As can been seen, sofosbuvir differs from the compound of presentExample 2, in that it possesses a beta-methyl group at the 2′-position,whereas the compounds of the invention have a beta-chloro substituent atthis position. In the Fission phase III clinical trials reported inLawitz et al., N. Eng. J. Med., 2013; 368:1878-87, “Response rates inthe sofosbuvir ribavirin group were lower among patients with genotype 3infection than amongst those with genotype 2 infection (56% vs. 97%)”.

The antiviral activity of commercially available sofosbuvir and thecompound of Example 2 were compared in a genotype 3a transient repliconassay described in Kylefjord et al., J Virol. Methods 2014 195:156-63.

The EC₅₀ of sofosbuvir against genotype 3a is 0.230 μM+/−0.067, n=11,compared to an EC₅₀ of 0.072 μM+/−0.024, n=9 for the compound of Example2. A threefold better potency for the compound of the invention relativeto sofosbuvir is expected to markedly improve viral response rates inthe clinic.

The several-fold improvement in potency of the compounds of theinvention relative to sofosbuvir was maintained in transient repliconsof genotype 3a bearing the troublesome S282T mutation (conferringresistance to the HCV nucleoside mericitabine) where sofosbuvir had anEC₅₀ of 0.48 μM (n=1) and the compound of Example 2 had an EC₅₀ of 0.13μM (n=1). Similarly, the L159F/L320F double mutant generated by exposureto the nucleoside mericitabine and conferring cross resistance tosofosbuvir (Tong et al 2013 J. Infect. Dis., 209 (5), 668-75) wasprepared in a genotype 3a transient replicon as described above inKylefjord et al. ibid. In the this double mutant, sofosbuvir had an EC₅₀of 0.190 (n=1) whereas the compound of Example 2 shows an EC₅₀ of 0.062(n=1).

The compound of Example 2 was further evaluated to assess the antiviralactivity against genotypes 1-6 of HCV, both wild type and a number ofclinically relevant mutant strains. The result of the evaluationtogether with the average EC₅₀ of the genotypes and the correspondingvalues for sofosbuvir are summarised in Tables 2 and 3.

TABLE 2 Wild Type HCV Assay sofosbuvir Cmpd of Ex. 2 HCV GT1b (stable)0.098 (n = 126) 0.045 (n = 63) HCV GT1b (transient) 0.081 (n = 31) 0.044(n = 22) HCV GT1a* 0.131 (n = 16) 0.050 (n = 16) HCV GT2a replicon 0.048(n = 2) 0.023 (n = 2) HCV GT2a virus 0.054 (n = 4) 0.017 (n = 3) HCVGT3a* 0.129 (n = 8) 0.046 (n = 8) HCV GT4a* 0.218 (n = 8) 0.059 (n = 8)HCV GT5a* 0.114 (n = 5) 0.044 (n = 8) HCV GT6a* 0.179 (n = 4) 0.058 (n =6) AVG EC₅₀: 0.117 +/− 0.019 0.043 +/− 0.005 (potency increase 1.0 2.7vs sofosbuvir) EC₅₀ data (all in μM) presented as geometric means exceptAVG where the EC₅₀ is presented as the arithmetic means +/− SEM.*Chimeric replicons containing stated GT NS5B genes in con1 background.References: Con1 (Lohmann et al 2003); H77 (Blight et al 2003); GT2a(Wakita et al 2005); GT3a (Kylefjord et al 2013); GT4-6 (Wong et al2012); L159F/L320F (Tong et al 2013).

TABLE 3 Mutants HCV Assay sofosbuvir Cmpd of Ex. 2 HCV GT1b S282T 0.741(n = 18) 0.298 (n = 9) FC vs WT 8.8 6.8 HCV GT1b L159F/L320F 0.199 (n =5) 0.070 (n = 5) FC vs WT 2.5 1.6 HCV GT1a* S282T  1.01 (n = 5) 0.301 (n= 5) FC vs WT 7.7 6.0 HCV GT3a* S282T 0.521 (n = 6) 0.122 (n = 6) FC vsWT 4.0 2.7 HCV GT3a* L159F/L320F 0.190 (n = 1) 0.062 (n = 1) FC vs WT1.5 1.3 AVG EC₅₀ 0.532 +/− 0.158 0.171 +/− 0.054 (potency increase 1.03.1 vs sofosbuvir) EC₅₀ data (all in μM) presented as geometric meansexcept AVG where the EC₅₀ is presented as the arithmetic means +/− SEM.*Chimeric replicons containing stated GT NS5B genes in con1 background.References: Con1 (Lohmann et al 2003); H77 (Blight et al 2003); GT2a(Wakita et al 2005); GT3a (Kylefjord et al 2013); GT4-6 (Wong et al2012); L159F/L320F (Tong et al 2013).

From these two tables it is evident that the compound of present Example2 has a significant improved potency as compared to sofosbuvir againstHCV GT3a both in the wild type strain and in two clinically relevantmutant strains, while keeping the good potency against the othergenotypes.

Triphosphate Formation Assay

To estimate the ability of the compounds of the invention to generatethe antivirally active triphosphate species, a triphosphate formationassay was conducted. Each compound was tested in triplicates in theassay.

Fresh human plated hepatocytes (Biopredic, France) in 12-well plateswere used. Each well was plated with 0.76×10⁶ cells and incubated with a10 μM DMSO solution of compound (0.1% DMSO) in 1 mL incubation medium ina CO₂ incubator at 37° C. for 6-8 hours. The incubation was stopped bywashing each well with 1 mL ice cold Hank's balanced solution, pH 7.2twice, followed by addition of 0.5 mL ice cold 70% methanol. Immediatelyafter the addition of methanol, the cell-layer was detached from thebottom of the well by a cell scraper and sucked up and down 5-6 timeswith an automatic pipet. The cell suspension was transferred to a glassvial and stored over night at −20° C.

The samples, each consisting of various levels of protide, freenucleoside, and mono-, di- and triphosphate were then vortexed andcentrifuged at 10° C. for 10 minutes, at 14000 rpm in an Eppendorfcentrifuge 5417R. The supernatants were transferred to 2 mL glass vialswith insert and subjected to bioanalysis.

Bioanalysis

An internal standard (Indinavir) was added to each sample and thesamples (10 μL injection volume) were analysed on a two column systemcoupled to a QTRAP 5000 mass spectrometer. The two column systemconsisted of two binary pumps, X and Y, two switching valves and anautosampler. The two HPLC columns used were a Synergy POLAR-RP 50*4.6mm, 4 μm particles and a BioBasic AX 50*2.1 mm 5 μm particles. The LCflow rates were 0.4-0.6 mL/min mL/min (the higher flow rate were used inthe recondition step).

The HPLC mobile phases for the POLAR-RP column consisted of 10 mmol/Lammonium acetate in 2% acetonitrile (mobile phase A) and 10 mmol/Lammonium acetate in 90% acetonitrile (mobile phase B) and for theBioBasic AX column 10 mmol/L ammonium acetate in 2% acetonitrile (mobilephase C) and 1% ammonium hydroxide in 2% acetonitrile (mobile phase D).The HPLC gradient for pump Y started at 0% mobile phase B and was heldfor 2 min. During loading phase, the mobile phase went through thePOLAR-RP and BioBasic AX column, and prodrug, nucleoside and internalstandard were trapped on the POLAR-RP column: whereas the nucleotides(mono-, di- and triphosphates) eluted on to the BioBasic AX column andwere trapped there.

In the next step, the flow was switched from the POLAR-RP column to theMS and the mobile phase C switched from pump X to the BioBasic AXcolumn. The compounds on the POLAR-RP column were eluted with a gradientfrom 0% B up to 100% B in about two minutes and analyzed in positive ornegative mode using the multiple reaction monitoring mode (MRM).

In the last step the flow from the BioBasic AX column was switched tothe MS and the phosphates were eluted with a of about 7 minutes gradientup 50% D) and analyzed in positive or negative mode using MRM. Duringthe last step both columns are reconditioned.

Triphosphate concentration for each compound was then determined bycomparison with standard curves. The standard curves were made byanalysis of standard samples with known concentrations of triphosphate.The standards were ran in the same matrices as the test samples. Due tovariations in phosphorylation levels depending on hepatocyte donor, aninternal reference compound is required in each run of the assay inorder to enable ranking the results from different runs to each other.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

All documents referred to herein, including patents and patentapplications, are incorporated by reference in their entirety.

The invention claimed is:
 1. A compound represented by formula I:


2. A process for the preparation of a compound of claim 1 comprising thestep of reacting a diastereomenically pure compound of formula 1e withuracil, followed by removal of the hydroxy protecting groups as outlinedin the scheme:

wherein Pg¹ and Pg² are, the same or different, hydroxy protectinggroups selected from triisopropylsilyl, acetyl, benzoyl, p-chlorobenzoylor trityl; Lg is a leaving group, selected from methylsulfonate, ahalide or a phosphate ester.
 3. The process of claim 2, wherein Pg¹ andPg² both are triisopropylsilyl or benzoyl.
 4. The process of claim 3,wherein Pg¹ and Pg² both are triisopropylsilyl.
 5. The process of claim2, wherein Lg is methylsulfonate.
 6. The process of claim 2, wherein thereaction of compound 1e with uracil in step f is performed in thepresence of hexamethyldisilazane and a Lewis acid.
 7. The process ofclaim 6, wherein the Lewis acid is TMS triflate.
 8. The process of claim2, wherein removal the hydroxy protecting groups in step g is performedby treatment with an acid in an aqueous solution.
 9. The process ofclaim 2, wherein removal the hydroxy protecting groups is performed bytreatment with aqueous acetic acid.
 10. A diastereomerically purecompound of the formula:


11. A compound of the formula: